99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4205,"string":"4,205"}}},{"rowIdx":559,"cells":{"question":{"kind":"string","value":"Who gets more severe disease from MERS?"},"answer":{"kind":"string","value":"among older adults, especially males, with pre-existing diseases. "},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4206,"string":"4,206"}}},{"rowIdx":560,"cells":{"question":{"kind":"string","value":"What is the spread of MERS-CoV among humans, associated with?"},"answer":{"kind":"string","value":"with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs)."},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4207,"string":"4,207"}}},{"rowIdx":561,"cells":{"question":{"kind":"string","value":"What do the DCs suffer with MERS-CoV infection?"},"answer":{"kind":"string","value":" the equivalent of a 'common cold' from MERS-CoV infection,"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4208,"string":"4,208"}}},{"rowIdx":562,"cells":{"question":{"kind":"string","value":"What happens to humans infected by MERS-CoV virus?"},"answer":{"kind":"string","value":" the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases."},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4209,"string":"4,209"}}},{"rowIdx":563,"cells":{"question":{"kind":"string","value":"What is the incubation period of MERS?"},"answer":{"kind":"string","value":" five to six days, ranging from two to 16 days"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4210,"string":"4,210"}}},{"rowIdx":564,"cells":{"question":{"kind":"string","value":"What is the duration between when illness begins in one person and subsequently spreads to another?"},"answer":{"kind":"string","value":"13 to 14 days"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4211,"string":"4,211"}}},{"rowIdx":565,"cells":{"question":{"kind":"string","value":"What is the median time to death in case of progressive illness?"},"answer":{"kind":"string","value":"11 to 13 days"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4212,"string":"4,212"}}},{"rowIdx":566,"cells":{"question":{"kind":"string","value":"What is the progression of symptoms to disease?"},"answer":{"kind":"string","value":"Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome "},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4214,"string":"4,214"}}},{"rowIdx":567,"cells":{"question":{"kind":"string","value":"What did the later WHO definition of MERS clearly state?"},"answer":{"kind":"string","value":"a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms."},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4216,"string":"4,216"}}},{"rowIdx":568,"cells":{"question":{"kind":"string","value":"What percentage of cases KSA has been a source of?"},"answer":{"kind":"string","value":" 79 % "},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4217,"string":"4,217"}}},{"rowIdx":569,"cells":{"question":{"kind":"string","value":"What is severe MARS noted for?"},"answer":{"kind":"string","value":"its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions "},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4218,"string":"4,218"}}},{"rowIdx":570,"cells":{"question":{"kind":"string","value":"What symptoms appear among the confirmed cases of MERS?"},"answer":{"kind":"string","value":"fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4222,"string":"4,222"}}},{"rowIdx":571,"cells":{"question":{"kind":"string","value":"What do patients often present to a hospital with, in cases of MERS?"},"answer":{"kind":"string","value":" pneumonia"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4223,"string":"4,223"}}},{"rowIdx":572,"cells":{"question":{"kind":"string","value":"What can MERS disease progress to?"},"answer":{"kind":"string","value":"acute respiratory distress syndrome and multiorgan system failure"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4224,"string":"4,224"}}},{"rowIdx":573,"cells":{"question":{"kind":"string","value":"What percentage of all reported cases has MERS reportedly killed ?"},"answer":{"kind":"string","value":" 35 % "},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4225,"string":"4,225"}}},{"rowIdx":574,"cells":{"question":{"kind":"string","value":"What percentage of of all reported cases has MERS reportedly killed in KSA?"},"answer":{"kind":"string","value":"42 %"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4226,"string":"4,226"}}},{"rowIdx":575,"cells":{"question":{"kind":"string","value":"What was mortality in South Korea from MERS disease?"},"answer":{"kind":"string","value":"ranged from 7 % among younger age groups to 40 % among those aged 60 years and above"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4227,"string":"4,227"}}},{"rowIdx":576,"cells":{"question":{"kind":"string","value":"Which are the preferred method for MERS-CoV detection?"},"answer":{"kind":"string","value":" PCR-based techniques "},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4228,"string":"4,228"}}},{"rowIdx":577,"cells":{"question":{"kind":"string","value":"What have become a key diagnostic and taxonomic target for CoV species identification?"},"answer":{"kind":"string","value":"The first open reading frames "},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4229,"string":"4,229"}}},{"rowIdx":578,"cells":{"question":{"kind":"string","value":"Why it can be concluded that MERS-COV is a novel and distinct virus?"},"answer":{"kind":"string","value":" less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4230,"string":"4,230"}}},{"rowIdx":579,"cells":{"question":{"kind":"string","value":"What indicates the likely presence of infectious virus?"},"answer":{"kind":"string","value":" Detection of viral proteins rather than viral RNA"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4231,"string":"4,231"}}},{"rowIdx":580,"cells":{"question":{"kind":"string","value":"What is the sensitivity with which immunochromatographic tool could detect recombinant MERS-CoV nucleocapsid protein?"},"answer":{"kind":"string","value":"94 %"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4232,"string":"4,232"}}},{"rowIdx":581,"cells":{"question":{"kind":"string","value":"What is the specificity with which immunochromatographic tool could detect recombinant MERS-CoV nucleocapsid protein?"},"answer":{"kind":"string","value":"100 % "},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4233,"string":"4,233"}}},{"rowIdx":582,"cells":{"question":{"kind":"string","value":"What is a different approach for the detection?"},"answer":{"kind":"string","value":"monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4234,"string":"4,234"}}},{"rowIdx":583,"cells":{"question":{"kind":"string","value":"What is usual in serology testing?"},"answer":{"kind":"string","value":" to detect a viral footprint, in the form of antibodies"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4235,"string":"4,235"}}},{"rowIdx":584,"cells":{"question":{"kind":"string","value":"How have some sero-assays bypassed the risks of working with infectious virus?"},"answer":{"kind":"string","value":" by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins ["},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4238,"string":"4,238"}}},{"rowIdx":585,"cells":{"question":{"kind":"string","value":"How have some sero-assays bypassed the risks of working with infectious virus?"},"answer":{"kind":"string","value":"using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4239,"string":"4,239"}}},{"rowIdx":586,"cells":{"question":{"kind":"string","value":"What is the detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by?"},"answer":{"kind":"string","value":"by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) or MNT test."},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4240,"string":"4,240"}}},{"rowIdx":587,"cells":{"question":{"kind":"string","value":"What does the confirmatory process aim to ensure?"},"answer":{"kind":"string","value":"the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV)."},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4241,"string":"4,241"}}},{"rowIdx":588,"cells":{"question":{"kind":"string","value":"When does generally MERS infection does not trigger a detectable immune response?"},"answer":{"kind":"string","value":"if only mild disease or asymptomatic infection results"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4242,"string":"4,242"}}},{"rowIdx":589,"cells":{"question":{"kind":"string","value":"When does the WHO recommend samlinf from the LRT?"},"answer":{"kind":"string","value":" when sample collection is delayed by a week or more after onset of symptoms"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4243,"string":"4,243"}}},{"rowIdx":590,"cells":{"question":{"kind":"string","value":"What do the recommended samples include?"},"answer":{"kind":"string","value":"are recommended when URT sampling is to be conducted"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4244,"string":"4,244"}}},{"rowIdx":591,"cells":{"question":{"kind":"string","value":"What are recommended when URT sampling is to be conducted?"},"answer":{"kind":"string","value":" an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4245,"string":"4,245"}}},{"rowIdx":592,"cells":{"question":{"kind":"string","value":"What paired sera are preferable?"},"answer":{"kind":"string","value":" collected two to three weeks apart"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4246,"string":"4,246"}}},{"rowIdx":593,"cells":{"question":{"kind":"string","value":" When is a single sample suggested to be sufficient?"},"answer":{"kind":"string","value":"Human urine and stool have been found to contain MERS-CoV RNA"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4247,"string":"4,247"}}},{"rowIdx":594,"cells":{"question":{"kind":"string","value":"How long human urine and stool have been found to contain MERS-CoV RNA?"},"answer":{"kind":"string","value":"12 to 26 days after symptom onset "},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4248,"string":"4,248"}}},{"rowIdx":595,"cells":{"question":{"kind":"string","value":"What do individual studies report on viral shedding?"},"answer":{"kind":"string","value":" long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms."},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4249,"string":"4,249"}}},{"rowIdx":596,"cells":{"question":{"kind":"string","value":"What fraction of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) ?"},"answer":{"kind":"string","value":"Over three quarters of"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4262,"string":"4,262"}}},{"rowIdx":597,"cells":{"question":{"kind":"string","value":"How long MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum)? "},"answer":{"kind":"string","value":"at least 30 days"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4263,"string":"4,263"}}},{"rowIdx":598,"cells":{"question":{"kind":"string","value":"What percentage of contacts were still shedding RNA in their URT specimens?"},"answer":{"kind":"string","value":"30 %"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The\"\n]"},"document_id":{"kind":"number","value":1741,"string":"1,741"},"id":{"kind":"number","value":4264,"string":"4,264"}}},{"rowIdx":599,"cells":{"question":{"kind":"string","value":"What samples returned the highest MERS viral load values?"},"answer":{"kind":"string","value":" the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or"},"context_chunks":{"kind":"list like","value":["The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.","Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.","Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.","Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.","The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .","As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .","Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.","In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .","It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.","The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .","Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .","Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .","Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .","Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .","Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.","FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.","Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.","The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.","The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.","Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .","The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.","Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .","Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .","Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .","Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.","Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .","10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.","A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .","It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .","In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.","MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .","LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .","The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .","The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .","Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .","Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.","In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .","The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .","However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.","RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .","In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.","In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .","3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".","However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .","A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .","The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.","Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .","Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.","However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .","Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .","Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .","Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.","Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .","The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .","There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .","Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .","It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .","Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .","In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?","Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .","The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .","Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.","BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .","Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .","Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.","A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .","In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.","Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.","The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.","It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.","That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.","Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.","A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.","Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.","Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .","In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.","Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.","Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .","Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .","To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .","If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.","Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.","To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.","A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.","The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.","6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .","Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .","However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.","Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .","In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.","Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .","The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.","South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .","He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .","Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.","All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .","No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .","The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.","Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.","Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.","Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.","Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.","Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.","The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.","Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"],"string":"[\n \"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.\",\n \"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.\",\n \"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.\",\n \"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.\",\n \"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .\",\n \"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .\",\n \"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.\",\n \"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .\",\n \"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.\",\n \"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .\",\n \"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .\",\n \"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .\",\n \"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .\",\n \"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .\",\n \"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.\",\n \"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.\",\n \"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.\",\n \"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.\",\n \"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.\",\n \"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .\",\n \"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.\",\n \"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .\",\n \"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .\",\n \"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .\",\n \"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.\",\n \"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .\",\n \"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.\",\n \"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .\",\n \"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .\",\n \"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.\",\n \"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .\",\n \"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .\",\n \"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .\",\n \"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .\",\n \"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .\",\n \"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.\",\n \"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .\",\n \"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .\",\n \"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.\",\n \"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .\",\n \"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.\",\n \"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .\",\n \"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\".\",\n \"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \\\"storm in a teacup\\\". It is the low transmission rate that has prevented worldwide spread, despite many \\\"opportunities\\\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .\",\n \"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .\",\n \"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.\",\n \"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \\\"contact\\\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .\",\n \"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.\",\n \"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .\",\n \"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .\",\n \"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .\",\n \"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.\",\n \"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .\",\n \"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .\",\n \"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .\",\n \"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .\",\n \"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .\",\n \"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .\",\n \"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?\",\n \"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .\",\n \"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .\",\n \"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.\",\n \"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .\",\n \"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .\",\n \"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.\",\n \"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .\",\n \"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.\",\n \"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.\",\n \"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.\",\n \"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.\",\n \"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.\",\n \"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.\",\n \"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.\",\n \"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.\",\n \"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .\",\n \"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.\",\n \"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \\\"deep\\\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.\",\n \"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .\",\n \"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .\",\n \"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .\",\n \"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.\",\n \"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.\",\n \"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.\",\n \"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.\",\n \"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.\",\n \"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .\",\n \"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .\",\n \"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.\",\n \"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .\",\n \"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.\",\n \"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .\",\n \"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.\",\n \"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .\",\n \"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \\\"hospital shopping\\\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .\",\n \"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.\",\n \"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .\",\n \"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .\",\n \"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.\",\n \"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.\",\n \"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.\",\n \"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.\",\n \"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.\",\n \"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.\",\n \"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.\",\n \"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . 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How are Mammarenaviruses spread from rodents to humans? | mucosal exposure to aerosols or by direct contact of abraded skin with infectious material | [
"Several mammalian arenaviruses mammarenaviruses cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus LCMV , is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors.",
"Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.",
"We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. Text: Introduction Mammarenaviruses Arenaviridae: Mammarenavirus cause chronic infections of rodents worldwide . Invasion of human dwellings by infected rodents can result in human infections through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW .",
"Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW . The OW Lassa virus LASV , causative agent of Lassa fever LF , is the most significant OW mammarenaviral pathogen. LASV is estimated to infect several hundred thousand individuals annually in Western Africa, resulting in a high number of LF cases associated with high morbidity and lethality. Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever .",
"Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever . The worldwide-distributed OW mammarenavirus lymphocytic choriomeningitis virus LCMV is a neglected human pathogen of clinical significance especially in congenital infections . Moreover, LCMV poses a particular threat to immunocompromised individuals, as has been illustrated by fatal cases of LCMV infection associated with organ transplants . However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina.",
"However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina. Likewise, current anti-mammarenavirus therapy is limited to an offlabel use of the nucleoside analogue ribavirin that is only partially effective and can cause significant side effects . Development of effective anti-mammarenavirus drugs has been hampered by the lack of detailed understanding of virus/host-cell interactions required for mammarenavirus multiplication that could represent amenable targets for antiviral therapy. the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B .",
"the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B . To facilitate the identification of specific PPI between NP and host cell proteins, we used our mammarenavirus tri-segmented r3 platform to design an r3LCMV expressing a C-terminus Strep-tag version of enhanced green fluorescent protein r3LCMV/eGFP-Strep that we used as a negative control Fig 1A and 1B . We rescued rLCMV/Strep-NP and r3LCMV/eGFP-Strep and confirmed the expression of strep-tagged NP and eGFP in rLCMV/ Strep-NP-and r3LCMV/eGFP-Strep-infected cells, respectively Fig 1C . Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV.",
"Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV. However, both rLCMV/Strep-NP and r3LCMV/eGFP-Strep had WT-like growth kinetics and reached high titers. As with WT LCMV, infection with rLCMV/Strep-NP prevented production of bioactive IFN-I by cells in response to Sendai virus SeV infection as determined using an IFN bioassay based on protection against the cytopathic effect CPE induced by infection with vesicular stomatitis virus VSV Fig 1E . Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE.",
"Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE. We selected the human A549 cell line because lung epithelial cells are one of the initial cell targets of humans following inhalation of mammarenavirions. We infected A549 cells multiplicity of infection MOI = 0.1 with either rLCMV/Strep-NP or r3LCMV/eGFP-Strep Fig 2A . At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining.",
"At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining. We compared the pattern of stained protein bands detected between rLCMV/Strep-NP-and r3LCMV/eGFP-Strepinfected samples and confirmed the presence of Strep-NP and eGFP-Strep in pertinent samples Fig 2B . Protein complexes in the rest of eluates from the PD samples were concentrated by trichloroacetic acid TCA precipitation and subjected to trypsin digestion Fig 2A . Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 .",
"Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 . Filtering the data using these criteria resulted in identification of 139 candidate host-cell proteins as NP-interacting partners. Among 53 proteins found present in both NP-and eGFP-PD samples, 36 had spectral counts in the NP-PD sample that were less than two-fold higher than their corresponding spectral counts in the eGFP-PD sample Fig 2C and S1 Table . Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV.",
"Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV. The protein annotation through evolutionary relationship PANTHER protein family classification Biological Process of the NP-interacting protein candidates revealed that a large number of proteins were involved in metabolic and cellular processes Fig 2D . We also analyzed the molecular functions of the NP-interacting protein candidates according to the PAN-THER protein profile classification Fig 2E , which revealed diversified biochemical functions enriched for nucleic acid-binding proteins and chaperones. To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene .",
"To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene . OTPs are latest generation of siRNAs and offer significant advantages over previous generations. Off-target effects are primarily driven by antisense strand microRNA miR -like seed activity. In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy.",
"In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy. Numerous host-cell factors showed an anti-LCMV effect increased ZsG expression by kd of the genes , including microtubule-associated protein 1B MAP1B , dengue virus 2 DENV-2 , and chikungunya virus CHIKV . To begin assessing the potential biological implications of the identified NP-host cell protein interactions, we selected ATP1A1 and PHB given the availability of reagents, existing knowledge about their roles in cell physiology, and evidence of participation in multiplication of other viruses. We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers.",
"We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers. For this, we transfected A549 cells with siRNA targeting Expression of Strep-tagged proteins. A549 cells seeded 5.0 x 10 5 cells/well in a 6-well plate and cultured overnight were infected MOI = 0.1 with the indicated rLCMVs. At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins.",
"At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins. BHK-21 1.75 x 10 5 cells/well , A549 1.25 x 10 5 cells/well , or Vero E6 1.25 x 10 5 cells/well cells seeded in 24-well plates and cultured overnight were infected MOI = 0.01 with the indicated rLCMVs. At the indicated times pi, TCSs were collected and virus titers determined by IFFA. Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP.",
"Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP. A549 cells were infected MOI = 0.1 with the indicated rLCMV or mock-infected, and 36 h later infected with SeV MOI = 1 . At 24 h pi with SeV, TCS were collected and used, following virus inactivation by U.V., to treat Vero E6 cells for 16 h, followed by infection with rVSV MOI = 0.1 rVSV + or mockinfection rVSV - . At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production.",
"At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production. ATP1A1 or with NC siRNA 72 h prior to infection with rLCMV/ZsG. We found that siRNAmediated kd of ATP1A1 dramatically inhibited ZsGreen expression Fig 3Ci , which was associated with a significant reduction of infectious LCMV progeny Fig 3Cii . Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both.",
"Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both. Another consideration is that only a minor fraction of NP might be engaged in the interaction with a given host cell protein, and therefore, detection of these interactions would require highly sensitive methods such as LC-MS/MS. To overcome this problem we used confocal microscopy to examine the co-localization of NP with ATP1A1 and PHB in LCMV-infected cells. Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries .",
"Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries . The PHB inhibitor rocaglamide is a flavagline from an Aglaia tree used in traditional Chinese medicine that has potent anticancer activity . To examine whether pharmacological inhibition of ATP1A1 or PHB inhibited LCMV multiplication, we pretreated human A549 and HEK 293T , nonhuman primate Vero E6 , and rodent murine L929 and hamster BHK-21 cells with ouabain or rocaglamide and infected them with rLCMV/eGFP S1 Fig . Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition .",
"Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition . Likewise, we observed a dose-dependent rocaglamide inhibition of eGFP expression in all cell lines infected with rLCMV/eGFP S1B Fig . Consistent with these findings, production of infectious LCMV progeny was reduced by treatment with either ouabain or rocaglamide Fig 5A within a concentration range that had minimal impact on cell viability Fig 5B . To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability.",
"To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability. To gain insights about the mechanisms by which ouabain and rocaglamide exert their anti-LCMV activity, we examined effects of these compounds on distinct steps of the LCMV life cycle. First, we asked whether ouabain and rocaglamide affected cell entry of LCMV. We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection.",
"We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection. The timing of compound addition did not significantly change the number of eGFP-positive cells, indicating that neither ouabain nor rocaglamide inhibited cell entry of LCMV. The number of eGFP + cells in ouabain-treated cells was reduced at all time-of-addition points compared to vehicle dimethyl sulfoxide DMSO -treated cells, but was similar to that observed in ammonium chloride-treated cells. Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription.",
"Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription. To further investigate the effect of ouabain and rocaglamide on virus RNA synthesis, we infected A549 cells with a recombinant single-cycle infectious LCMV expressing eGFP rLCMVΔGPC/eGFP and treated cells with either ouabain or rocaglamide. Seventy-two hours later, we determined percent normalized eGFP expression in infected cells Fig 6B . Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis.",
"Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis. We also examined the effect of ouabain and rocaglamide on the late step of the arenavirus life cycle, Z-mediated budding. For this experiment, we transfected cells with Z-Strep-and Z-FLAG DYKDDDDK epitope -expressing plasmids from LCMV and LASV, respectively. At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide.",
"At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide. At 24 h, we determined by WB levels of Z protein in both whole cell lysates and associated with virus-like particles VLPs collected from TCS. Treatment with rocaglamide, but not with ouabain, caused a reduction in both LCMV and LASV Z budding efficiency Fig 6C and 6D . The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments.",
"The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments. The first experiment involved the use of a newly generated single-cycle infectious recombinant LCMV expressing the reporter gene ZsGreen scrLCMV/ZsG-P2A-NP to infect MOI = 0.1 A549 cells 1 st infection . These cells were subsequently transfected with a plasmid expressing LCMV GPC. After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F .",
"After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F . The second experiment involved infection MOI = 0.1 of cells with WT LCMV, and 48 h later we washed infected cells three times to remove the extracellular infectious progeny produced during the first 48 h of infection. Then, fresh media containing ouabain or DMSO vehicle control were added, and 24 h later we determined virus titers in TCS Fig 6G . Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections.",
"Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections. Since ouabain and rocaglamide inhibited different steps of the LCMV life cycle, we examined whether their use in combination results in a synergistic anti-LCMV effect. For this experiment, we infected A549 cells with rLCMV/eGFP and treated them with ouabain and rocaglamide using different concentrations and combinations. At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide.",
"At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide. We next asked whether the ATP1A1 and PHB host-cell factors contributed also to multiplication of viral hemorrhagic fever-causing LASV. We treated A549 cells with ouabain, bufalin, or rocaglamide and inoculated the treated cells with recombinant LASV expressing eGFP rLASV/eGFP . eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 .",
"eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 . We also tested the effect of compounds targeting ATP1A1 and PHB on multiplication of JUNV. Consistent with our results obtained with LCMV and LASV, ouabain, bufalin, and rocaglamide strongly suppressed JUNV multiplication S3 Fig . These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses.",
"These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses. Our approach using a recombinant LCMV expressing NP with an affinity tag facilitated defining the NP interactome in the context of LCMV infection. Recently, using an NP-specific monoclonal antibody mAb to precipitate NP and associated cellular protein partners in a mammarenavirus NP interactome, King et al. identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al.",
"identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al. Differences in both experimental conditions and analysis methodologies used to generate the LCMV NP interactome documented by King et al. and ours likely h post-transfection, cells were washed with fresh media to eliminate Z-mediated production of VLPs in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain or Roc-A at the indicated concentrations. VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D .",
"VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D . Budding efficiency for each sample was estimated by dividing the signal intensity of the Z protein associated with VLPs by that of Z detected in the cell lysate. Numbers on the bottom of panel C correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments.",
"Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments. F Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected MOI = 0.1 with scrLCMV/ZsG 1 st infection for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared.",
"At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. ZsGreen signal intensity was measured by a fluorescent plate reader. GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%.",
"GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%. Data represent means ± SD from three independent experiments. contributed to the observed differences between data sets. Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems.",
"Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems. We used results of the eGFP-Strep interactome, determined in r3LCMV/eGFP-Strep-infected cells, as a control to filter out non-specific NP interactions, which may have resulted in a higher degree of stringency than in the study by King et al for selection of NP-interacting candidates. The combined information provided by the NP interactome reported by King et al. and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction .",
"and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction . The anti-IFN activity of NP was mapped to its C-terminal part and linked to the 3'-5' exonuclease domain present with the NP C-terminal part . Inhibitor-B kinase ε IKKε was identified as an NP-binding protein using plasmid-mediated overexpression in transfected cells , and the NP-IKKε binding affinity correlated with NP's ability to inhibit IFN-I induction . We, as well as the work by King et al. , did not detect this NP-IKKε interaction.",
"We, as well as the work by King et al. , did not detect this NP-IKKε interaction. This discrepancy may have been caused by very low expression of IKKε in LCMV-infected cells, which prevented detection of IKKε by LC-MS/MS. Alternatively, NP-IKKε interaction could possibility be temporarily regulated and take place at early times pi, but could be mostly absent at 48 h pi, the time at which we prepared the cell lysates for our proteomics studies. Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit .",
"Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit . ATP1A1 represents one of four α subunits . Recent evidence has suggested that the Na + /K + -ATPase is involved in multiple cell signaling pathways that are independent of its ion-pumping function . Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase .",
"Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase . Thus, a low concentration of ouabain induces a conformational change in ATP1A1 that results in activation and release of proto-oncogene tyrosine protein kinase, Src, from ATP1A1, followed by activation of as yet unknown downstream signaling that inhibits, for instance, cell entry of murine hepatitis virus MHV . However, our results indicated that ouabain did not interfere with LCMV cell entry. In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV.",
"In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV. Moreover, cell entry of JUNV occurs also by clathrin-mediated endocytosis , a process affected by Src signaling. Ouabain has been clinically used in several European countries for the management of congestive heart failure, whereas bufalin has been tested in clinical trials for cancer treatments , and the CST digoxin has been FDA-approved since 1997 to treat heart failure and atrial fibrillation. Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry.",
"Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry. In contrast, PHB was reported to be a cell entry receptor for DENV-2 and CHIKV . On the other hand, PHB did not act as a virus cell entry receptor for HCV. Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV .",
"Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV . These findings indicate that PHB is involved in different steps of the life cycle of a variety of viruses, and thereby an attractive target for the development of broad-spectrum antiviral drugs. Rocaglate is a group of natural compounds, which includes rocaglamide, that inhibits protein synthesis by targeting the ATP-dependent DEAD-box RNA helicase eukaryotic initiation factor 4A eIF4A and exerts anti-tumor activity . The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication.",
"The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication. These proteins, which included MAP1B, might have anti-LCMV activity. MAP1B has been shown to bind to nonstructural proteins 1 NS1 and 2 NS2 of human respiratory syncytial virus HRSV . NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined .",
"NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined . Examining the role of NP-MAP1B interaction in modulating NP's ability to inhibit induction of type I IFN is of interest. We identified among the NP-interacting host cell proteins the RNA helicase Moloney leukemia virus 10 MOV10 , which has been reported to be an antiviral factor for FLUAV , retroviruses , and DENV-2 . We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions.",
"We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions. MOV10 was shown to enhance IRF-3-mediated IFN-I induction following SeV infection through a tank binding kinase 1 TBK1 -independent and IKKε-dependent manner. This finding was further supported by demonstrating MOV10-IKKε interaction by co-immunoprecipitation studies . We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined.",
"We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined. Several members of the mammalian chaperonin-containing T-complex CCT were identified as prominent hits in our NP interactome. The mammalian CCT is critical for folding of many proteins with important functions in diverse cellular processes , and may protect complex protein topologies within its central cavity during biosynthesis and folding . The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV .",
"The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV . However, the role of these CCT proteins in virus multiplication remains unknown and may involve functions other than acting as molecular chaperones. Previous studies documented the presence of several components of the of eIF4F, including 4A, within viral replication-transcription complexes RTC detected in cells infected with LCMV and TCRV . These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity.",
"These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity. In this work, we documented the generation of rLCMV/Strep-NP and its use to define the NP-interactome in infected cells. We presented evidence that ATP1A1 and PHB contribute to efficient multiplication of mammarenaviruses using genetics and pharmacological inhibition of the genes. Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins.",
"Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins. Future studies elucidating the roles of pro-and antiviral host-cell factors identified in this study in mammarenavirus multiplication will advance our understanding of the multiple functions of NP and uncover novel cellular targets for the development of antimammarenaviral drugs. In addition, by identifying proviral host-cell factors, drugs that are already approved can be repurposing as therapeutics to combat human pathogenic mammarenavirus infections. Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described .",
"Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described . Generation of rLCMV/NP D382A and SeV, strain Cantell, was described 30, 82, 83 . An rLCMV lacking GPC and expressing eGFP rLCMVΔGPC/eGFP was generated by reverse genetics using procedures previously described . rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B .",
"rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B . The rLCMV expressing eGFP rLCMV/eGFP was generated as described , and the rLCMV expressing ZsGreen rLCMV/ZsG instead of eGFP was generated by reverse genetics using similar procedures to generate rLCMV/eGFP. Generation of rLASV expressing eGFP rLASV/eGFP will be described elsewhere. A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG.",
"A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG. scrLCMV/ZsG-P2A-NP was rescued by reverse genetics using similar procedures to generate rLCMVΔGPC/eGFP . LCMV titers were determined by immunofocus forming assay IFFA as described . Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA .",
"Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA . VSV titers were determined by a plaque assay. Total cell lysates were prepared in PD lysis buffer + 250 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% TritonX-100, 10% glycerol, 1 mM of MgCl 2 , 1 μM of CaCl 2 , 1 μM of ZnCl 2 and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA .",
"Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA . To detect Strep-tagged proteins, membranes were reacted with mouse monoclonal antibodies to Strep QIAGEN, Germantown, MD , eGFP Takara Bio USA, Mountain View, CA , GP 2 We33/ 36 , ATP1A1 TehrmoFisher Scientific, Rockford, IL , PHB Abcam, Cambridge, MA or rabbit polyclonal antibodies to α-tubulin Cell Signaling Technologies, Danvers, MA or glyceraldehyde-3-phosphate dehydrogenase GAPDH, Millipore , respectively, followed by incubation with appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G IgG antibodies Jackson ImmunoResearch Laboratories, West Grove, PA . SuperSignal West Pico or Femto chemiluminescent substrate Thermo Fisher Scientific was used to elicit chemiluminescent signals that were visualized using ImageQuant LAS 4000 Imager GE Healthcare Bio-Sciences, Pittsburgh, PA . Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1.",
"Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1. At 48 h pi, cells were washed three times with ice-cold PBS, scraped into fresh ice-cold PBS, and centrifuged at 400 x g at 4˚C for 10 min. Supernatant was removed, and cells were lysed with 12 ml of PD lysis buffer + supplemented with halt protease and phosphatase inhibitor cocktail Thermo Fisher Scientific and 5 μg/ml of deoxyribonuclease I Worthington Biochemical Corporation, Lakewood, NJ . Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C.",
"Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C. After 2 h of incubation, the resin was washed three times with PD lysis buffer + and once with PD lysis buffer without TritonX-100 PD lysis buffer - . After the centrifugation at 1,600 x g and 4˚C for 5 min, the last wash buffer was removed, and protein complexes associated with the resin were eluted into 2 ml of PD lysis buffer - containing 2.5 mM of desthiobiotin. The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn.",
"The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn. A MudPIT microcolumn was prepared by first creating a Kasil frit at one end of an un-deactivated 250-μm outside diameter OD capillary tube interior diameter of 360 μm Agilent Technologies, Inc., Santa Clara, CA . The Kasil frit was prepared by briefly dipping a 20-30-cm capillary tube in 300 μl of Kasil 1624 potassium silicate well-mixed solution PQ Corporation, Malvern, PA and 100 μl of formamide, curing at 100˚C for 4 h, and cutting the frit to a length of %2 mm. Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading.",
"Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading. MudPIT analysis. An analytical reversed-phase liquid chromatography column was generated by pulling a 100-μm interior diameter ID of 360 μm OD capillary tube Polymicro Technologies, Phoenix, AZ to 5-μm ID tip. Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid .",
"Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid . MudPIT and analytical columns were assembled using a zero-dead volume union Upchurch Scientific, Oak Harbor, WA . LC-MS/MS analysis was performed with an Agilent high-pressure LC pump Agilent and linear quadrupole ion dual cell trap Orbitrap Velos Thermo using an in-house built electrospray stage. Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient.",
"Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient. Data analysis. Protein and peptide identification were performed with Integrated Proteomics Pipeline-IP2 Integrated Proteomics Applications, San Diego, CA. integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in.",
"integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in. Spectrum raw files were extracted into ms2 files from raw files using open source RawExtract 1.9.9 Scripps Research Institute, La Jolla, CA; and the tandem mass spectra were searched against a human protein database UniprotKB . To accurately estimate peptide probabilities and false discovery rates, we used a decoy database containing the reversed sequences of all the proteins appended to the target database. Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system.",
"Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system. The search space included half and fully tryptic peptide candidates that fell within the mass tolerance window with no miscleavage constraint. Carbamidomethylation +57.02146 Da of cysteine was considered as a static modification. siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG.",
"siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG. siRNA target host-cell proteins were selected based on availability of validated siRNA sequences. The siRNAs we used to examine the effects on LCMV multiplication of knockdown expression of NP-interacting host cell protein candidate hits corresponded to the Genome-wide ON TAR-GET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene; Dharmacon, Lafayette, CO . A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min.",
"A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. The total protein concentration of clarified cell lysate was measured by Pierce BCA Protein Assay Kit Thermo Fisher Scientific . The same amount of protein from each sample was subjected to SDS-PAGE, and the protein expression of siRNA-targeted genes was analyzed by western blots. Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI .",
"Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI . Green fluorescence eGFP or ZsGreen and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, Bio-Tek, Winooski, VT . Mock-and virus-infected cells were fixed with 4% PFA. After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488.",
"After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488. In some samples, primary antibody against ATP1A1 or PHB was omitted to determine background fluorescence. To visualize nuclei, DAPI Fluoromount-G SouthernBiotech, Birmingham, AL was used to mount coverslips on a slide glass. Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss .",
"Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss . Eight green NP-positive cells were marked and every pixel in the marked area was plotted in the scatter diagram based on its intensity level from each channel. Thresholds for green and red channels were determined using mock-infected cells stained with VL-4-AF488 anti-NP and anti-mouse IgG antibody conjugated with Alexa Fluor 568, without using anti-ATP1A1 or -PHB antibodies. Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels.",
"Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels. This calculation was repeated for eight individual cells. To assess the specificity of co-localization, we determined weighted CC by taking into consideration the brightness of each channel signal. Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software.",
"Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software. A549 or Vero E6 cells seeded 2.0 x 10 4 cells/well in a 96-well plate and cultured overnight were treated with 3-fold serial compound dilutions at 37˚C and 5% CO 2 for 2 h, followed by infection with rLCMV/eGFP MOI = 0.01 . Compounds were present to study endpoint. At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%.",
"At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%. The IC 50 concentrations were determined using GraphPad Prism. A549 or Vero E6 cells seeded in a 96-well plate 2.0 x 10 4 cells/well and cultured overnight were treated with 3-fold serial compound dilutions and cultured at 37˚C and 5% CO 2 for 48 h. Then, CellTiter 96 AQ ueous one solution reagent Promega, Madison, WI was added. Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%.",
"Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%. The CC 50 concentrations were determined using GraphPad Prism. A plasmid expressing C-terminus Strep-tagged Z protein pC-LCMV-Z-Strep was generated using similar procedure to generate a plasmid expressing C-terminus FLAG-tagged LASV Z protein pC-LASV-Z-FLAG , and the budding assay was performed as previously described . Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium.",
"Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium. After the removal of the last wash medium, cells were cultured in fresh medium containing ouabain 30 or 40 nM or rocaglamide 50 or 100 nM or equivalent concentration of DMSO, and 24 h later, virion-like particle VLP -containing TCS and cells were collected. Total cell lysate was prepared by lysing the cells with lysis buffer 1% NP-40, 50 mM of Tris-HCl pH 8.0 , 62.5 mM NaCl, 0.4% sodium deoxycholate . After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots.",
"After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots. A549 cells infected with rLCMV/eGFP were harvested using Accutase cell detachment solution Innovative Cell Technologies, San Diego, CA and fixed with 4% PFA in PBS. eGFP expression was examined by flow cytometry using a BD LSR II Becton Dickson , and data were analyzed with FlowJo Tree Star, Inc., Ashland, OR . 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO .",
"293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO . At 48 h pi, TCS was collected and used to infect fresh monolayers of BHK-21 cells seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . A549 cells seeded 2 x 10 5 cells/well in a 96-well plate and cultured overnight were treated with combinations of different concentrations of ouabain and rocaglamide for 2 h and then infected MOI = 0.01 with rLCMV/eGFP. Compounds were present in the culture medium throughout the experiment. At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . eGFP readouts were normalized by DAPI readouts, and normalized data were used to analyze synergistic effect of the two compounds by the MacSynergy II program . Data were analyzed for p values by a two-tailed unpaired t test using GraphPad Prism software."
] | 1,687 | 5,149 |
What are the main groups for Mammarenaviruses? | Old World (OW) and New World (NW) | [
"Several mammalian arenaviruses mammarenaviruses cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus LCMV , is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors.",
"Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.",
"We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. Text: Introduction Mammarenaviruses Arenaviridae: Mammarenavirus cause chronic infections of rodents worldwide . Invasion of human dwellings by infected rodents can result in human infections through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW .",
"Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW . The OW Lassa virus LASV , causative agent of Lassa fever LF , is the most significant OW mammarenaviral pathogen. LASV is estimated to infect several hundred thousand individuals annually in Western Africa, resulting in a high number of LF cases associated with high morbidity and lethality. Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever .",
"Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever . The worldwide-distributed OW mammarenavirus lymphocytic choriomeningitis virus LCMV is a neglected human pathogen of clinical significance especially in congenital infections . Moreover, LCMV poses a particular threat to immunocompromised individuals, as has been illustrated by fatal cases of LCMV infection associated with organ transplants . However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina.",
"However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina. Likewise, current anti-mammarenavirus therapy is limited to an offlabel use of the nucleoside analogue ribavirin that is only partially effective and can cause significant side effects . Development of effective anti-mammarenavirus drugs has been hampered by the lack of detailed understanding of virus/host-cell interactions required for mammarenavirus multiplication that could represent amenable targets for antiviral therapy. the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B .",
"the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B . To facilitate the identification of specific PPI between NP and host cell proteins, we used our mammarenavirus tri-segmented r3 platform to design an r3LCMV expressing a C-terminus Strep-tag version of enhanced green fluorescent protein r3LCMV/eGFP-Strep that we used as a negative control Fig 1A and 1B . We rescued rLCMV/Strep-NP and r3LCMV/eGFP-Strep and confirmed the expression of strep-tagged NP and eGFP in rLCMV/ Strep-NP-and r3LCMV/eGFP-Strep-infected cells, respectively Fig 1C . Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV.",
"Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV. However, both rLCMV/Strep-NP and r3LCMV/eGFP-Strep had WT-like growth kinetics and reached high titers. As with WT LCMV, infection with rLCMV/Strep-NP prevented production of bioactive IFN-I by cells in response to Sendai virus SeV infection as determined using an IFN bioassay based on protection against the cytopathic effect CPE induced by infection with vesicular stomatitis virus VSV Fig 1E . Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE.",
"Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE. We selected the human A549 cell line because lung epithelial cells are one of the initial cell targets of humans following inhalation of mammarenavirions. We infected A549 cells multiplicity of infection MOI = 0.1 with either rLCMV/Strep-NP or r3LCMV/eGFP-Strep Fig 2A . At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining.",
"At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining. We compared the pattern of stained protein bands detected between rLCMV/Strep-NP-and r3LCMV/eGFP-Strepinfected samples and confirmed the presence of Strep-NP and eGFP-Strep in pertinent samples Fig 2B . Protein complexes in the rest of eluates from the PD samples were concentrated by trichloroacetic acid TCA precipitation and subjected to trypsin digestion Fig 2A . Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 .",
"Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 . Filtering the data using these criteria resulted in identification of 139 candidate host-cell proteins as NP-interacting partners. Among 53 proteins found present in both NP-and eGFP-PD samples, 36 had spectral counts in the NP-PD sample that were less than two-fold higher than their corresponding spectral counts in the eGFP-PD sample Fig 2C and S1 Table . Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV.",
"Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV. The protein annotation through evolutionary relationship PANTHER protein family classification Biological Process of the NP-interacting protein candidates revealed that a large number of proteins were involved in metabolic and cellular processes Fig 2D . We also analyzed the molecular functions of the NP-interacting protein candidates according to the PAN-THER protein profile classification Fig 2E , which revealed diversified biochemical functions enriched for nucleic acid-binding proteins and chaperones. To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene .",
"To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene . OTPs are latest generation of siRNAs and offer significant advantages over previous generations. Off-target effects are primarily driven by antisense strand microRNA miR -like seed activity. In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy.",
"In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy. Numerous host-cell factors showed an anti-LCMV effect increased ZsG expression by kd of the genes , including microtubule-associated protein 1B MAP1B , dengue virus 2 DENV-2 , and chikungunya virus CHIKV . To begin assessing the potential biological implications of the identified NP-host cell protein interactions, we selected ATP1A1 and PHB given the availability of reagents, existing knowledge about their roles in cell physiology, and evidence of participation in multiplication of other viruses. We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers.",
"We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers. For this, we transfected A549 cells with siRNA targeting Expression of Strep-tagged proteins. A549 cells seeded 5.0 x 10 5 cells/well in a 6-well plate and cultured overnight were infected MOI = 0.1 with the indicated rLCMVs. At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins.",
"At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins. BHK-21 1.75 x 10 5 cells/well , A549 1.25 x 10 5 cells/well , or Vero E6 1.25 x 10 5 cells/well cells seeded in 24-well plates and cultured overnight were infected MOI = 0.01 with the indicated rLCMVs. At the indicated times pi, TCSs were collected and virus titers determined by IFFA. Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP.",
"Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP. A549 cells were infected MOI = 0.1 with the indicated rLCMV or mock-infected, and 36 h later infected with SeV MOI = 1 . At 24 h pi with SeV, TCS were collected and used, following virus inactivation by U.V., to treat Vero E6 cells for 16 h, followed by infection with rVSV MOI = 0.1 rVSV + or mockinfection rVSV - . At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production.",
"At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production. ATP1A1 or with NC siRNA 72 h prior to infection with rLCMV/ZsG. We found that siRNAmediated kd of ATP1A1 dramatically inhibited ZsGreen expression Fig 3Ci , which was associated with a significant reduction of infectious LCMV progeny Fig 3Cii . Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both.",
"Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both. Another consideration is that only a minor fraction of NP might be engaged in the interaction with a given host cell protein, and therefore, detection of these interactions would require highly sensitive methods such as LC-MS/MS. To overcome this problem we used confocal microscopy to examine the co-localization of NP with ATP1A1 and PHB in LCMV-infected cells. Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries .",
"Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries . The PHB inhibitor rocaglamide is a flavagline from an Aglaia tree used in traditional Chinese medicine that has potent anticancer activity . To examine whether pharmacological inhibition of ATP1A1 or PHB inhibited LCMV multiplication, we pretreated human A549 and HEK 293T , nonhuman primate Vero E6 , and rodent murine L929 and hamster BHK-21 cells with ouabain or rocaglamide and infected them with rLCMV/eGFP S1 Fig . Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition .",
"Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition . Likewise, we observed a dose-dependent rocaglamide inhibition of eGFP expression in all cell lines infected with rLCMV/eGFP S1B Fig . Consistent with these findings, production of infectious LCMV progeny was reduced by treatment with either ouabain or rocaglamide Fig 5A within a concentration range that had minimal impact on cell viability Fig 5B . To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability.",
"To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability. To gain insights about the mechanisms by which ouabain and rocaglamide exert their anti-LCMV activity, we examined effects of these compounds on distinct steps of the LCMV life cycle. First, we asked whether ouabain and rocaglamide affected cell entry of LCMV. We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection.",
"We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection. The timing of compound addition did not significantly change the number of eGFP-positive cells, indicating that neither ouabain nor rocaglamide inhibited cell entry of LCMV. The number of eGFP + cells in ouabain-treated cells was reduced at all time-of-addition points compared to vehicle dimethyl sulfoxide DMSO -treated cells, but was similar to that observed in ammonium chloride-treated cells. Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription.",
"Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription. To further investigate the effect of ouabain and rocaglamide on virus RNA synthesis, we infected A549 cells with a recombinant single-cycle infectious LCMV expressing eGFP rLCMVΔGPC/eGFP and treated cells with either ouabain or rocaglamide. Seventy-two hours later, we determined percent normalized eGFP expression in infected cells Fig 6B . Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis.",
"Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis. We also examined the effect of ouabain and rocaglamide on the late step of the arenavirus life cycle, Z-mediated budding. For this experiment, we transfected cells with Z-Strep-and Z-FLAG DYKDDDDK epitope -expressing plasmids from LCMV and LASV, respectively. At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide.",
"At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide. At 24 h, we determined by WB levels of Z protein in both whole cell lysates and associated with virus-like particles VLPs collected from TCS. Treatment with rocaglamide, but not with ouabain, caused a reduction in both LCMV and LASV Z budding efficiency Fig 6C and 6D . The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments.",
"The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments. The first experiment involved the use of a newly generated single-cycle infectious recombinant LCMV expressing the reporter gene ZsGreen scrLCMV/ZsG-P2A-NP to infect MOI = 0.1 A549 cells 1 st infection . These cells were subsequently transfected with a plasmid expressing LCMV GPC. After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F .",
"After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F . The second experiment involved infection MOI = 0.1 of cells with WT LCMV, and 48 h later we washed infected cells three times to remove the extracellular infectious progeny produced during the first 48 h of infection. Then, fresh media containing ouabain or DMSO vehicle control were added, and 24 h later we determined virus titers in TCS Fig 6G . Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections.",
"Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections. Since ouabain and rocaglamide inhibited different steps of the LCMV life cycle, we examined whether their use in combination results in a synergistic anti-LCMV effect. For this experiment, we infected A549 cells with rLCMV/eGFP and treated them with ouabain and rocaglamide using different concentrations and combinations. At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide.",
"At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide. We next asked whether the ATP1A1 and PHB host-cell factors contributed also to multiplication of viral hemorrhagic fever-causing LASV. We treated A549 cells with ouabain, bufalin, or rocaglamide and inoculated the treated cells with recombinant LASV expressing eGFP rLASV/eGFP . eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 .",
"eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 . We also tested the effect of compounds targeting ATP1A1 and PHB on multiplication of JUNV. Consistent with our results obtained with LCMV and LASV, ouabain, bufalin, and rocaglamide strongly suppressed JUNV multiplication S3 Fig . These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses.",
"These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses. Our approach using a recombinant LCMV expressing NP with an affinity tag facilitated defining the NP interactome in the context of LCMV infection. Recently, using an NP-specific monoclonal antibody mAb to precipitate NP and associated cellular protein partners in a mammarenavirus NP interactome, King et al. identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al.",
"identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al. Differences in both experimental conditions and analysis methodologies used to generate the LCMV NP interactome documented by King et al. and ours likely h post-transfection, cells were washed with fresh media to eliminate Z-mediated production of VLPs in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain or Roc-A at the indicated concentrations. VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D .",
"VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D . Budding efficiency for each sample was estimated by dividing the signal intensity of the Z protein associated with VLPs by that of Z detected in the cell lysate. Numbers on the bottom of panel C correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments.",
"Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments. F Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected MOI = 0.1 with scrLCMV/ZsG 1 st infection for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared.",
"At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. ZsGreen signal intensity was measured by a fluorescent plate reader. GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%.",
"GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%. Data represent means ± SD from three independent experiments. contributed to the observed differences between data sets. Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems.",
"Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems. We used results of the eGFP-Strep interactome, determined in r3LCMV/eGFP-Strep-infected cells, as a control to filter out non-specific NP interactions, which may have resulted in a higher degree of stringency than in the study by King et al for selection of NP-interacting candidates. The combined information provided by the NP interactome reported by King et al. and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction .",
"and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction . The anti-IFN activity of NP was mapped to its C-terminal part and linked to the 3'-5' exonuclease domain present with the NP C-terminal part . Inhibitor-B kinase ε IKKε was identified as an NP-binding protein using plasmid-mediated overexpression in transfected cells , and the NP-IKKε binding affinity correlated with NP's ability to inhibit IFN-I induction . We, as well as the work by King et al. , did not detect this NP-IKKε interaction.",
"We, as well as the work by King et al. , did not detect this NP-IKKε interaction. This discrepancy may have been caused by very low expression of IKKε in LCMV-infected cells, which prevented detection of IKKε by LC-MS/MS. Alternatively, NP-IKKε interaction could possibility be temporarily regulated and take place at early times pi, but could be mostly absent at 48 h pi, the time at which we prepared the cell lysates for our proteomics studies. Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit .",
"Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit . ATP1A1 represents one of four α subunits . Recent evidence has suggested that the Na + /K + -ATPase is involved in multiple cell signaling pathways that are independent of its ion-pumping function . Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase .",
"Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase . Thus, a low concentration of ouabain induces a conformational change in ATP1A1 that results in activation and release of proto-oncogene tyrosine protein kinase, Src, from ATP1A1, followed by activation of as yet unknown downstream signaling that inhibits, for instance, cell entry of murine hepatitis virus MHV . However, our results indicated that ouabain did not interfere with LCMV cell entry. In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV.",
"In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV. Moreover, cell entry of JUNV occurs also by clathrin-mediated endocytosis , a process affected by Src signaling. Ouabain has been clinically used in several European countries for the management of congestive heart failure, whereas bufalin has been tested in clinical trials for cancer treatments , and the CST digoxin has been FDA-approved since 1997 to treat heart failure and atrial fibrillation. Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry.",
"Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry. In contrast, PHB was reported to be a cell entry receptor for DENV-2 and CHIKV . On the other hand, PHB did not act as a virus cell entry receptor for HCV. Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV .",
"Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV . These findings indicate that PHB is involved in different steps of the life cycle of a variety of viruses, and thereby an attractive target for the development of broad-spectrum antiviral drugs. Rocaglate is a group of natural compounds, which includes rocaglamide, that inhibits protein synthesis by targeting the ATP-dependent DEAD-box RNA helicase eukaryotic initiation factor 4A eIF4A and exerts anti-tumor activity . The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication.",
"The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication. These proteins, which included MAP1B, might have anti-LCMV activity. MAP1B has been shown to bind to nonstructural proteins 1 NS1 and 2 NS2 of human respiratory syncytial virus HRSV . NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined .",
"NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined . Examining the role of NP-MAP1B interaction in modulating NP's ability to inhibit induction of type I IFN is of interest. We identified among the NP-interacting host cell proteins the RNA helicase Moloney leukemia virus 10 MOV10 , which has been reported to be an antiviral factor for FLUAV , retroviruses , and DENV-2 . We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions.",
"We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions. MOV10 was shown to enhance IRF-3-mediated IFN-I induction following SeV infection through a tank binding kinase 1 TBK1 -independent and IKKε-dependent manner. This finding was further supported by demonstrating MOV10-IKKε interaction by co-immunoprecipitation studies . We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined.",
"We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined. Several members of the mammalian chaperonin-containing T-complex CCT were identified as prominent hits in our NP interactome. The mammalian CCT is critical for folding of many proteins with important functions in diverse cellular processes , and may protect complex protein topologies within its central cavity during biosynthesis and folding . The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV .",
"The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV . However, the role of these CCT proteins in virus multiplication remains unknown and may involve functions other than acting as molecular chaperones. Previous studies documented the presence of several components of the of eIF4F, including 4A, within viral replication-transcription complexes RTC detected in cells infected with LCMV and TCRV . These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity.",
"These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity. In this work, we documented the generation of rLCMV/Strep-NP and its use to define the NP-interactome in infected cells. We presented evidence that ATP1A1 and PHB contribute to efficient multiplication of mammarenaviruses using genetics and pharmacological inhibition of the genes. Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins.",
"Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins. Future studies elucidating the roles of pro-and antiviral host-cell factors identified in this study in mammarenavirus multiplication will advance our understanding of the multiple functions of NP and uncover novel cellular targets for the development of antimammarenaviral drugs. In addition, by identifying proviral host-cell factors, drugs that are already approved can be repurposing as therapeutics to combat human pathogenic mammarenavirus infections. Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described .",
"Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described . Generation of rLCMV/NP D382A and SeV, strain Cantell, was described 30, 82, 83 . An rLCMV lacking GPC and expressing eGFP rLCMVΔGPC/eGFP was generated by reverse genetics using procedures previously described . rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B .",
"rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B . The rLCMV expressing eGFP rLCMV/eGFP was generated as described , and the rLCMV expressing ZsGreen rLCMV/ZsG instead of eGFP was generated by reverse genetics using similar procedures to generate rLCMV/eGFP. Generation of rLASV expressing eGFP rLASV/eGFP will be described elsewhere. A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG.",
"A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG. scrLCMV/ZsG-P2A-NP was rescued by reverse genetics using similar procedures to generate rLCMVΔGPC/eGFP . LCMV titers were determined by immunofocus forming assay IFFA as described . Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA .",
"Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA . VSV titers were determined by a plaque assay. Total cell lysates were prepared in PD lysis buffer + 250 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% TritonX-100, 10% glycerol, 1 mM of MgCl 2 , 1 μM of CaCl 2 , 1 μM of ZnCl 2 and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA .",
"Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA . To detect Strep-tagged proteins, membranes were reacted with mouse monoclonal antibodies to Strep QIAGEN, Germantown, MD , eGFP Takara Bio USA, Mountain View, CA , GP 2 We33/ 36 , ATP1A1 TehrmoFisher Scientific, Rockford, IL , PHB Abcam, Cambridge, MA or rabbit polyclonal antibodies to α-tubulin Cell Signaling Technologies, Danvers, MA or glyceraldehyde-3-phosphate dehydrogenase GAPDH, Millipore , respectively, followed by incubation with appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G IgG antibodies Jackson ImmunoResearch Laboratories, West Grove, PA . SuperSignal West Pico or Femto chemiluminescent substrate Thermo Fisher Scientific was used to elicit chemiluminescent signals that were visualized using ImageQuant LAS 4000 Imager GE Healthcare Bio-Sciences, Pittsburgh, PA . Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1.",
"Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1. At 48 h pi, cells were washed three times with ice-cold PBS, scraped into fresh ice-cold PBS, and centrifuged at 400 x g at 4˚C for 10 min. Supernatant was removed, and cells were lysed with 12 ml of PD lysis buffer + supplemented with halt protease and phosphatase inhibitor cocktail Thermo Fisher Scientific and 5 μg/ml of deoxyribonuclease I Worthington Biochemical Corporation, Lakewood, NJ . Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C.",
"Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C. After 2 h of incubation, the resin was washed three times with PD lysis buffer + and once with PD lysis buffer without TritonX-100 PD lysis buffer - . After the centrifugation at 1,600 x g and 4˚C for 5 min, the last wash buffer was removed, and protein complexes associated with the resin were eluted into 2 ml of PD lysis buffer - containing 2.5 mM of desthiobiotin. The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn.",
"The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn. A MudPIT microcolumn was prepared by first creating a Kasil frit at one end of an un-deactivated 250-μm outside diameter OD capillary tube interior diameter of 360 μm Agilent Technologies, Inc., Santa Clara, CA . The Kasil frit was prepared by briefly dipping a 20-30-cm capillary tube in 300 μl of Kasil 1624 potassium silicate well-mixed solution PQ Corporation, Malvern, PA and 100 μl of formamide, curing at 100˚C for 4 h, and cutting the frit to a length of %2 mm. Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading.",
"Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading. MudPIT analysis. An analytical reversed-phase liquid chromatography column was generated by pulling a 100-μm interior diameter ID of 360 μm OD capillary tube Polymicro Technologies, Phoenix, AZ to 5-μm ID tip. Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid .",
"Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid . MudPIT and analytical columns were assembled using a zero-dead volume union Upchurch Scientific, Oak Harbor, WA . LC-MS/MS analysis was performed with an Agilent high-pressure LC pump Agilent and linear quadrupole ion dual cell trap Orbitrap Velos Thermo using an in-house built electrospray stage. Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient.",
"Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient. Data analysis. Protein and peptide identification were performed with Integrated Proteomics Pipeline-IP2 Integrated Proteomics Applications, San Diego, CA. integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in.",
"integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in. Spectrum raw files were extracted into ms2 files from raw files using open source RawExtract 1.9.9 Scripps Research Institute, La Jolla, CA; and the tandem mass spectra were searched against a human protein database UniprotKB . To accurately estimate peptide probabilities and false discovery rates, we used a decoy database containing the reversed sequences of all the proteins appended to the target database. Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system.",
"Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system. The search space included half and fully tryptic peptide candidates that fell within the mass tolerance window with no miscleavage constraint. Carbamidomethylation +57.02146 Da of cysteine was considered as a static modification. siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG.",
"siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG. siRNA target host-cell proteins were selected based on availability of validated siRNA sequences. The siRNAs we used to examine the effects on LCMV multiplication of knockdown expression of NP-interacting host cell protein candidate hits corresponded to the Genome-wide ON TAR-GET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene; Dharmacon, Lafayette, CO . A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min.",
"A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. The total protein concentration of clarified cell lysate was measured by Pierce BCA Protein Assay Kit Thermo Fisher Scientific . The same amount of protein from each sample was subjected to SDS-PAGE, and the protein expression of siRNA-targeted genes was analyzed by western blots. Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI .",
"Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI . Green fluorescence eGFP or ZsGreen and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, Bio-Tek, Winooski, VT . Mock-and virus-infected cells were fixed with 4% PFA. After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488.",
"After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488. In some samples, primary antibody against ATP1A1 or PHB was omitted to determine background fluorescence. To visualize nuclei, DAPI Fluoromount-G SouthernBiotech, Birmingham, AL was used to mount coverslips on a slide glass. Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss .",
"Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss . Eight green NP-positive cells were marked and every pixel in the marked area was plotted in the scatter diagram based on its intensity level from each channel. Thresholds for green and red channels were determined using mock-infected cells stained with VL-4-AF488 anti-NP and anti-mouse IgG antibody conjugated with Alexa Fluor 568, without using anti-ATP1A1 or -PHB antibodies. Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels.",
"Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels. This calculation was repeated for eight individual cells. To assess the specificity of co-localization, we determined weighted CC by taking into consideration the brightness of each channel signal. Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software.",
"Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software. A549 or Vero E6 cells seeded 2.0 x 10 4 cells/well in a 96-well plate and cultured overnight were treated with 3-fold serial compound dilutions at 37˚C and 5% CO 2 for 2 h, followed by infection with rLCMV/eGFP MOI = 0.01 . Compounds were present to study endpoint. At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%.",
"At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%. The IC 50 concentrations were determined using GraphPad Prism. A549 or Vero E6 cells seeded in a 96-well plate 2.0 x 10 4 cells/well and cultured overnight were treated with 3-fold serial compound dilutions and cultured at 37˚C and 5% CO 2 for 48 h. Then, CellTiter 96 AQ ueous one solution reagent Promega, Madison, WI was added. Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%.",
"Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%. The CC 50 concentrations were determined using GraphPad Prism. A plasmid expressing C-terminus Strep-tagged Z protein pC-LCMV-Z-Strep was generated using similar procedure to generate a plasmid expressing C-terminus FLAG-tagged LASV Z protein pC-LASV-Z-FLAG , and the budding assay was performed as previously described . Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium.",
"Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium. After the removal of the last wash medium, cells were cultured in fresh medium containing ouabain 30 or 40 nM or rocaglamide 50 or 100 nM or equivalent concentration of DMSO, and 24 h later, virion-like particle VLP -containing TCS and cells were collected. Total cell lysate was prepared by lysing the cells with lysis buffer 1% NP-40, 50 mM of Tris-HCl pH 8.0 , 62.5 mM NaCl, 0.4% sodium deoxycholate . After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots.",
"After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots. A549 cells infected with rLCMV/eGFP were harvested using Accutase cell detachment solution Innovative Cell Technologies, San Diego, CA and fixed with 4% PFA in PBS. eGFP expression was examined by flow cytometry using a BD LSR II Becton Dickson , and data were analyzed with FlowJo Tree Star, Inc., Ashland, OR . 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO .",
"293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO . At 48 h pi, TCS was collected and used to infect fresh monolayers of BHK-21 cells seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . A549 cells seeded 2 x 10 5 cells/well in a 96-well plate and cultured overnight were treated with combinations of different concentrations of ouabain and rocaglamide for 2 h and then infected MOI = 0.01 with rLCMV/eGFP. Compounds were present in the culture medium throughout the experiment. At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . eGFP readouts were normalized by DAPI readouts, and normalized data were used to analyze synergistic effect of the two compounds by the MacSynergy II program . Data were analyzed for p values by a two-tailed unpaired t test using GraphPad Prism software."
] | 1,687 | 5,150 |
What is the cause of Lassa fever? | OW Lassa virus (LASV) | [
"Several mammalian arenaviruses mammarenaviruses cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus LCMV , is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors.",
"Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.",
"We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. Text: Introduction Mammarenaviruses Arenaviridae: Mammarenavirus cause chronic infections of rodents worldwide . Invasion of human dwellings by infected rodents can result in human infections through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW .",
"Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW . The OW Lassa virus LASV , causative agent of Lassa fever LF , is the most significant OW mammarenaviral pathogen. LASV is estimated to infect several hundred thousand individuals annually in Western Africa, resulting in a high number of LF cases associated with high morbidity and lethality. Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever .",
"Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever . The worldwide-distributed OW mammarenavirus lymphocytic choriomeningitis virus LCMV is a neglected human pathogen of clinical significance especially in congenital infections . Moreover, LCMV poses a particular threat to immunocompromised individuals, as has been illustrated by fatal cases of LCMV infection associated with organ transplants . However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina.",
"However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina. Likewise, current anti-mammarenavirus therapy is limited to an offlabel use of the nucleoside analogue ribavirin that is only partially effective and can cause significant side effects . Development of effective anti-mammarenavirus drugs has been hampered by the lack of detailed understanding of virus/host-cell interactions required for mammarenavirus multiplication that could represent amenable targets for antiviral therapy. the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B .",
"the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B . To facilitate the identification of specific PPI between NP and host cell proteins, we used our mammarenavirus tri-segmented r3 platform to design an r3LCMV expressing a C-terminus Strep-tag version of enhanced green fluorescent protein r3LCMV/eGFP-Strep that we used as a negative control Fig 1A and 1B . We rescued rLCMV/Strep-NP and r3LCMV/eGFP-Strep and confirmed the expression of strep-tagged NP and eGFP in rLCMV/ Strep-NP-and r3LCMV/eGFP-Strep-infected cells, respectively Fig 1C . Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV.",
"Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV. However, both rLCMV/Strep-NP and r3LCMV/eGFP-Strep had WT-like growth kinetics and reached high titers. As with WT LCMV, infection with rLCMV/Strep-NP prevented production of bioactive IFN-I by cells in response to Sendai virus SeV infection as determined using an IFN bioassay based on protection against the cytopathic effect CPE induced by infection with vesicular stomatitis virus VSV Fig 1E . Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE.",
"Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE. We selected the human A549 cell line because lung epithelial cells are one of the initial cell targets of humans following inhalation of mammarenavirions. We infected A549 cells multiplicity of infection MOI = 0.1 with either rLCMV/Strep-NP or r3LCMV/eGFP-Strep Fig 2A . At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining.",
"At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining. We compared the pattern of stained protein bands detected between rLCMV/Strep-NP-and r3LCMV/eGFP-Strepinfected samples and confirmed the presence of Strep-NP and eGFP-Strep in pertinent samples Fig 2B . Protein complexes in the rest of eluates from the PD samples were concentrated by trichloroacetic acid TCA precipitation and subjected to trypsin digestion Fig 2A . Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 .",
"Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 . Filtering the data using these criteria resulted in identification of 139 candidate host-cell proteins as NP-interacting partners. Among 53 proteins found present in both NP-and eGFP-PD samples, 36 had spectral counts in the NP-PD sample that were less than two-fold higher than their corresponding spectral counts in the eGFP-PD sample Fig 2C and S1 Table . Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV.",
"Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV. The protein annotation through evolutionary relationship PANTHER protein family classification Biological Process of the NP-interacting protein candidates revealed that a large number of proteins were involved in metabolic and cellular processes Fig 2D . We also analyzed the molecular functions of the NP-interacting protein candidates according to the PAN-THER protein profile classification Fig 2E , which revealed diversified biochemical functions enriched for nucleic acid-binding proteins and chaperones. To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene .",
"To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene . OTPs are latest generation of siRNAs and offer significant advantages over previous generations. Off-target effects are primarily driven by antisense strand microRNA miR -like seed activity. In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy.",
"In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy. Numerous host-cell factors showed an anti-LCMV effect increased ZsG expression by kd of the genes , including microtubule-associated protein 1B MAP1B , dengue virus 2 DENV-2 , and chikungunya virus CHIKV . To begin assessing the potential biological implications of the identified NP-host cell protein interactions, we selected ATP1A1 and PHB given the availability of reagents, existing knowledge about their roles in cell physiology, and evidence of participation in multiplication of other viruses. We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers.",
"We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers. For this, we transfected A549 cells with siRNA targeting Expression of Strep-tagged proteins. A549 cells seeded 5.0 x 10 5 cells/well in a 6-well plate and cultured overnight were infected MOI = 0.1 with the indicated rLCMVs. At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins.",
"At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins. BHK-21 1.75 x 10 5 cells/well , A549 1.25 x 10 5 cells/well , or Vero E6 1.25 x 10 5 cells/well cells seeded in 24-well plates and cultured overnight were infected MOI = 0.01 with the indicated rLCMVs. At the indicated times pi, TCSs were collected and virus titers determined by IFFA. Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP.",
"Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP. A549 cells were infected MOI = 0.1 with the indicated rLCMV or mock-infected, and 36 h later infected with SeV MOI = 1 . At 24 h pi with SeV, TCS were collected and used, following virus inactivation by U.V., to treat Vero E6 cells for 16 h, followed by infection with rVSV MOI = 0.1 rVSV + or mockinfection rVSV - . At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production.",
"At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production. ATP1A1 or with NC siRNA 72 h prior to infection with rLCMV/ZsG. We found that siRNAmediated kd of ATP1A1 dramatically inhibited ZsGreen expression Fig 3Ci , which was associated with a significant reduction of infectious LCMV progeny Fig 3Cii . Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both.",
"Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both. Another consideration is that only a minor fraction of NP might be engaged in the interaction with a given host cell protein, and therefore, detection of these interactions would require highly sensitive methods such as LC-MS/MS. To overcome this problem we used confocal microscopy to examine the co-localization of NP with ATP1A1 and PHB in LCMV-infected cells. Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries .",
"Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries . The PHB inhibitor rocaglamide is a flavagline from an Aglaia tree used in traditional Chinese medicine that has potent anticancer activity . To examine whether pharmacological inhibition of ATP1A1 or PHB inhibited LCMV multiplication, we pretreated human A549 and HEK 293T , nonhuman primate Vero E6 , and rodent murine L929 and hamster BHK-21 cells with ouabain or rocaglamide and infected them with rLCMV/eGFP S1 Fig . Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition .",
"Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition . Likewise, we observed a dose-dependent rocaglamide inhibition of eGFP expression in all cell lines infected with rLCMV/eGFP S1B Fig . Consistent with these findings, production of infectious LCMV progeny was reduced by treatment with either ouabain or rocaglamide Fig 5A within a concentration range that had minimal impact on cell viability Fig 5B . To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability.",
"To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability. To gain insights about the mechanisms by which ouabain and rocaglamide exert their anti-LCMV activity, we examined effects of these compounds on distinct steps of the LCMV life cycle. First, we asked whether ouabain and rocaglamide affected cell entry of LCMV. We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection.",
"We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection. The timing of compound addition did not significantly change the number of eGFP-positive cells, indicating that neither ouabain nor rocaglamide inhibited cell entry of LCMV. The number of eGFP + cells in ouabain-treated cells was reduced at all time-of-addition points compared to vehicle dimethyl sulfoxide DMSO -treated cells, but was similar to that observed in ammonium chloride-treated cells. Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription.",
"Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription. To further investigate the effect of ouabain and rocaglamide on virus RNA synthesis, we infected A549 cells with a recombinant single-cycle infectious LCMV expressing eGFP rLCMVΔGPC/eGFP and treated cells with either ouabain or rocaglamide. Seventy-two hours later, we determined percent normalized eGFP expression in infected cells Fig 6B . Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis.",
"Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis. We also examined the effect of ouabain and rocaglamide on the late step of the arenavirus life cycle, Z-mediated budding. For this experiment, we transfected cells with Z-Strep-and Z-FLAG DYKDDDDK epitope -expressing plasmids from LCMV and LASV, respectively. At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide.",
"At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide. At 24 h, we determined by WB levels of Z protein in both whole cell lysates and associated with virus-like particles VLPs collected from TCS. Treatment with rocaglamide, but not with ouabain, caused a reduction in both LCMV and LASV Z budding efficiency Fig 6C and 6D . The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments.",
"The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments. The first experiment involved the use of a newly generated single-cycle infectious recombinant LCMV expressing the reporter gene ZsGreen scrLCMV/ZsG-P2A-NP to infect MOI = 0.1 A549 cells 1 st infection . These cells were subsequently transfected with a plasmid expressing LCMV GPC. After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F .",
"After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F . The second experiment involved infection MOI = 0.1 of cells with WT LCMV, and 48 h later we washed infected cells three times to remove the extracellular infectious progeny produced during the first 48 h of infection. Then, fresh media containing ouabain or DMSO vehicle control were added, and 24 h later we determined virus titers in TCS Fig 6G . Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections.",
"Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections. Since ouabain and rocaglamide inhibited different steps of the LCMV life cycle, we examined whether their use in combination results in a synergistic anti-LCMV effect. For this experiment, we infected A549 cells with rLCMV/eGFP and treated them with ouabain and rocaglamide using different concentrations and combinations. At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide.",
"At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide. We next asked whether the ATP1A1 and PHB host-cell factors contributed also to multiplication of viral hemorrhagic fever-causing LASV. We treated A549 cells with ouabain, bufalin, or rocaglamide and inoculated the treated cells with recombinant LASV expressing eGFP rLASV/eGFP . eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 .",
"eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 . We also tested the effect of compounds targeting ATP1A1 and PHB on multiplication of JUNV. Consistent with our results obtained with LCMV and LASV, ouabain, bufalin, and rocaglamide strongly suppressed JUNV multiplication S3 Fig . These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses.",
"These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses. Our approach using a recombinant LCMV expressing NP with an affinity tag facilitated defining the NP interactome in the context of LCMV infection. Recently, using an NP-specific monoclonal antibody mAb to precipitate NP and associated cellular protein partners in a mammarenavirus NP interactome, King et al. identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al.",
"identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al. Differences in both experimental conditions and analysis methodologies used to generate the LCMV NP interactome documented by King et al. and ours likely h post-transfection, cells were washed with fresh media to eliminate Z-mediated production of VLPs in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain or Roc-A at the indicated concentrations. VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D .",
"VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D . Budding efficiency for each sample was estimated by dividing the signal intensity of the Z protein associated with VLPs by that of Z detected in the cell lysate. Numbers on the bottom of panel C correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments.",
"Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments. F Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected MOI = 0.1 with scrLCMV/ZsG 1 st infection for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared.",
"At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. ZsGreen signal intensity was measured by a fluorescent plate reader. GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%.",
"GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%. Data represent means ± SD from three independent experiments. contributed to the observed differences between data sets. Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems.",
"Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems. We used results of the eGFP-Strep interactome, determined in r3LCMV/eGFP-Strep-infected cells, as a control to filter out non-specific NP interactions, which may have resulted in a higher degree of stringency than in the study by King et al for selection of NP-interacting candidates. The combined information provided by the NP interactome reported by King et al. and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction .",
"and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction . The anti-IFN activity of NP was mapped to its C-terminal part and linked to the 3'-5' exonuclease domain present with the NP C-terminal part . Inhibitor-B kinase ε IKKε was identified as an NP-binding protein using plasmid-mediated overexpression in transfected cells , and the NP-IKKε binding affinity correlated with NP's ability to inhibit IFN-I induction . We, as well as the work by King et al. , did not detect this NP-IKKε interaction.",
"We, as well as the work by King et al. , did not detect this NP-IKKε interaction. This discrepancy may have been caused by very low expression of IKKε in LCMV-infected cells, which prevented detection of IKKε by LC-MS/MS. Alternatively, NP-IKKε interaction could possibility be temporarily regulated and take place at early times pi, but could be mostly absent at 48 h pi, the time at which we prepared the cell lysates for our proteomics studies. Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit .",
"Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit . ATP1A1 represents one of four α subunits . Recent evidence has suggested that the Na + /K + -ATPase is involved in multiple cell signaling pathways that are independent of its ion-pumping function . Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase .",
"Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase . Thus, a low concentration of ouabain induces a conformational change in ATP1A1 that results in activation and release of proto-oncogene tyrosine protein kinase, Src, from ATP1A1, followed by activation of as yet unknown downstream signaling that inhibits, for instance, cell entry of murine hepatitis virus MHV . However, our results indicated that ouabain did not interfere with LCMV cell entry. In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV.",
"In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV. Moreover, cell entry of JUNV occurs also by clathrin-mediated endocytosis , a process affected by Src signaling. Ouabain has been clinically used in several European countries for the management of congestive heart failure, whereas bufalin has been tested in clinical trials for cancer treatments , and the CST digoxin has been FDA-approved since 1997 to treat heart failure and atrial fibrillation. Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry.",
"Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry. In contrast, PHB was reported to be a cell entry receptor for DENV-2 and CHIKV . On the other hand, PHB did not act as a virus cell entry receptor for HCV. Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV .",
"Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV . These findings indicate that PHB is involved in different steps of the life cycle of a variety of viruses, and thereby an attractive target for the development of broad-spectrum antiviral drugs. Rocaglate is a group of natural compounds, which includes rocaglamide, that inhibits protein synthesis by targeting the ATP-dependent DEAD-box RNA helicase eukaryotic initiation factor 4A eIF4A and exerts anti-tumor activity . The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication.",
"The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication. These proteins, which included MAP1B, might have anti-LCMV activity. MAP1B has been shown to bind to nonstructural proteins 1 NS1 and 2 NS2 of human respiratory syncytial virus HRSV . NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined .",
"NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined . Examining the role of NP-MAP1B interaction in modulating NP's ability to inhibit induction of type I IFN is of interest. We identified among the NP-interacting host cell proteins the RNA helicase Moloney leukemia virus 10 MOV10 , which has been reported to be an antiviral factor for FLUAV , retroviruses , and DENV-2 . We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions.",
"We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions. MOV10 was shown to enhance IRF-3-mediated IFN-I induction following SeV infection through a tank binding kinase 1 TBK1 -independent and IKKε-dependent manner. This finding was further supported by demonstrating MOV10-IKKε interaction by co-immunoprecipitation studies . We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined.",
"We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined. Several members of the mammalian chaperonin-containing T-complex CCT were identified as prominent hits in our NP interactome. The mammalian CCT is critical for folding of many proteins with important functions in diverse cellular processes , and may protect complex protein topologies within its central cavity during biosynthesis and folding . The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV .",
"The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV . However, the role of these CCT proteins in virus multiplication remains unknown and may involve functions other than acting as molecular chaperones. Previous studies documented the presence of several components of the of eIF4F, including 4A, within viral replication-transcription complexes RTC detected in cells infected with LCMV and TCRV . These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity.",
"These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity. In this work, we documented the generation of rLCMV/Strep-NP and its use to define the NP-interactome in infected cells. We presented evidence that ATP1A1 and PHB contribute to efficient multiplication of mammarenaviruses using genetics and pharmacological inhibition of the genes. Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins.",
"Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins. Future studies elucidating the roles of pro-and antiviral host-cell factors identified in this study in mammarenavirus multiplication will advance our understanding of the multiple functions of NP and uncover novel cellular targets for the development of antimammarenaviral drugs. In addition, by identifying proviral host-cell factors, drugs that are already approved can be repurposing as therapeutics to combat human pathogenic mammarenavirus infections. Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described .",
"Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described . Generation of rLCMV/NP D382A and SeV, strain Cantell, was described 30, 82, 83 . An rLCMV lacking GPC and expressing eGFP rLCMVΔGPC/eGFP was generated by reverse genetics using procedures previously described . rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B .",
"rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B . The rLCMV expressing eGFP rLCMV/eGFP was generated as described , and the rLCMV expressing ZsGreen rLCMV/ZsG instead of eGFP was generated by reverse genetics using similar procedures to generate rLCMV/eGFP. Generation of rLASV expressing eGFP rLASV/eGFP will be described elsewhere. A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG.",
"A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG. scrLCMV/ZsG-P2A-NP was rescued by reverse genetics using similar procedures to generate rLCMVΔGPC/eGFP . LCMV titers were determined by immunofocus forming assay IFFA as described . Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA .",
"Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA . VSV titers were determined by a plaque assay. Total cell lysates were prepared in PD lysis buffer + 250 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% TritonX-100, 10% glycerol, 1 mM of MgCl 2 , 1 μM of CaCl 2 , 1 μM of ZnCl 2 and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA .",
"Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA . To detect Strep-tagged proteins, membranes were reacted with mouse monoclonal antibodies to Strep QIAGEN, Germantown, MD , eGFP Takara Bio USA, Mountain View, CA , GP 2 We33/ 36 , ATP1A1 TehrmoFisher Scientific, Rockford, IL , PHB Abcam, Cambridge, MA or rabbit polyclonal antibodies to α-tubulin Cell Signaling Technologies, Danvers, MA or glyceraldehyde-3-phosphate dehydrogenase GAPDH, Millipore , respectively, followed by incubation with appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G IgG antibodies Jackson ImmunoResearch Laboratories, West Grove, PA . SuperSignal West Pico or Femto chemiluminescent substrate Thermo Fisher Scientific was used to elicit chemiluminescent signals that were visualized using ImageQuant LAS 4000 Imager GE Healthcare Bio-Sciences, Pittsburgh, PA . Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1.",
"Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1. At 48 h pi, cells were washed three times with ice-cold PBS, scraped into fresh ice-cold PBS, and centrifuged at 400 x g at 4˚C for 10 min. Supernatant was removed, and cells were lysed with 12 ml of PD lysis buffer + supplemented with halt protease and phosphatase inhibitor cocktail Thermo Fisher Scientific and 5 μg/ml of deoxyribonuclease I Worthington Biochemical Corporation, Lakewood, NJ . Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C.",
"Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C. After 2 h of incubation, the resin was washed three times with PD lysis buffer + and once with PD lysis buffer without TritonX-100 PD lysis buffer - . After the centrifugation at 1,600 x g and 4˚C for 5 min, the last wash buffer was removed, and protein complexes associated with the resin were eluted into 2 ml of PD lysis buffer - containing 2.5 mM of desthiobiotin. The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn.",
"The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn. A MudPIT microcolumn was prepared by first creating a Kasil frit at one end of an un-deactivated 250-μm outside diameter OD capillary tube interior diameter of 360 μm Agilent Technologies, Inc., Santa Clara, CA . The Kasil frit was prepared by briefly dipping a 20-30-cm capillary tube in 300 μl of Kasil 1624 potassium silicate well-mixed solution PQ Corporation, Malvern, PA and 100 μl of formamide, curing at 100˚C for 4 h, and cutting the frit to a length of %2 mm. Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading.",
"Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading. MudPIT analysis. An analytical reversed-phase liquid chromatography column was generated by pulling a 100-μm interior diameter ID of 360 μm OD capillary tube Polymicro Technologies, Phoenix, AZ to 5-μm ID tip. Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid .",
"Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid . MudPIT and analytical columns were assembled using a zero-dead volume union Upchurch Scientific, Oak Harbor, WA . LC-MS/MS analysis was performed with an Agilent high-pressure LC pump Agilent and linear quadrupole ion dual cell trap Orbitrap Velos Thermo using an in-house built electrospray stage. Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient.",
"Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient. Data analysis. Protein and peptide identification were performed with Integrated Proteomics Pipeline-IP2 Integrated Proteomics Applications, San Diego, CA. integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in.",
"integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in. Spectrum raw files were extracted into ms2 files from raw files using open source RawExtract 1.9.9 Scripps Research Institute, La Jolla, CA; and the tandem mass spectra were searched against a human protein database UniprotKB . To accurately estimate peptide probabilities and false discovery rates, we used a decoy database containing the reversed sequences of all the proteins appended to the target database. Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system.",
"Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system. The search space included half and fully tryptic peptide candidates that fell within the mass tolerance window with no miscleavage constraint. Carbamidomethylation +57.02146 Da of cysteine was considered as a static modification. siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG.",
"siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG. siRNA target host-cell proteins were selected based on availability of validated siRNA sequences. The siRNAs we used to examine the effects on LCMV multiplication of knockdown expression of NP-interacting host cell protein candidate hits corresponded to the Genome-wide ON TAR-GET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene; Dharmacon, Lafayette, CO . A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min.",
"A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. The total protein concentration of clarified cell lysate was measured by Pierce BCA Protein Assay Kit Thermo Fisher Scientific . The same amount of protein from each sample was subjected to SDS-PAGE, and the protein expression of siRNA-targeted genes was analyzed by western blots. Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI .",
"Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI . Green fluorescence eGFP or ZsGreen and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, Bio-Tek, Winooski, VT . Mock-and virus-infected cells were fixed with 4% PFA. After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488.",
"After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488. In some samples, primary antibody against ATP1A1 or PHB was omitted to determine background fluorescence. To visualize nuclei, DAPI Fluoromount-G SouthernBiotech, Birmingham, AL was used to mount coverslips on a slide glass. Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss .",
"Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss . Eight green NP-positive cells were marked and every pixel in the marked area was plotted in the scatter diagram based on its intensity level from each channel. Thresholds for green and red channels were determined using mock-infected cells stained with VL-4-AF488 anti-NP and anti-mouse IgG antibody conjugated with Alexa Fluor 568, without using anti-ATP1A1 or -PHB antibodies. Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels.",
"Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels. This calculation was repeated for eight individual cells. To assess the specificity of co-localization, we determined weighted CC by taking into consideration the brightness of each channel signal. Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software.",
"Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software. A549 or Vero E6 cells seeded 2.0 x 10 4 cells/well in a 96-well plate and cultured overnight were treated with 3-fold serial compound dilutions at 37˚C and 5% CO 2 for 2 h, followed by infection with rLCMV/eGFP MOI = 0.01 . Compounds were present to study endpoint. At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%.",
"At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%. The IC 50 concentrations were determined using GraphPad Prism. A549 or Vero E6 cells seeded in a 96-well plate 2.0 x 10 4 cells/well and cultured overnight were treated with 3-fold serial compound dilutions and cultured at 37˚C and 5% CO 2 for 48 h. Then, CellTiter 96 AQ ueous one solution reagent Promega, Madison, WI was added. Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%.",
"Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%. The CC 50 concentrations were determined using GraphPad Prism. A plasmid expressing C-terminus Strep-tagged Z protein pC-LCMV-Z-Strep was generated using similar procedure to generate a plasmid expressing C-terminus FLAG-tagged LASV Z protein pC-LASV-Z-FLAG , and the budding assay was performed as previously described . Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium.",
"Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium. After the removal of the last wash medium, cells were cultured in fresh medium containing ouabain 30 or 40 nM or rocaglamide 50 or 100 nM or equivalent concentration of DMSO, and 24 h later, virion-like particle VLP -containing TCS and cells were collected. Total cell lysate was prepared by lysing the cells with lysis buffer 1% NP-40, 50 mM of Tris-HCl pH 8.0 , 62.5 mM NaCl, 0.4% sodium deoxycholate . After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots.",
"After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots. A549 cells infected with rLCMV/eGFP were harvested using Accutase cell detachment solution Innovative Cell Technologies, San Diego, CA and fixed with 4% PFA in PBS. eGFP expression was examined by flow cytometry using a BD LSR II Becton Dickson , and data were analyzed with FlowJo Tree Star, Inc., Ashland, OR . 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO .",
"293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO . At 48 h pi, TCS was collected and used to infect fresh monolayers of BHK-21 cells seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . A549 cells seeded 2 x 10 5 cells/well in a 96-well plate and cultured overnight were treated with combinations of different concentrations of ouabain and rocaglamide for 2 h and then infected MOI = 0.01 with rLCMV/eGFP. Compounds were present in the culture medium throughout the experiment. At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . eGFP readouts were normalized by DAPI readouts, and normalized data were used to analyze synergistic effect of the two compounds by the MacSynergy II program . Data were analyzed for p values by a two-tailed unpaired t test using GraphPad Prism software."
] | 1,687 | 5,151 |
What type of vaccine is JUNV, Candid#1? | live attenuated | [
"Several mammalian arenaviruses mammarenaviruses cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus LCMV , is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors.",
"Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.",
"We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. Text: Introduction Mammarenaviruses Arenaviridae: Mammarenavirus cause chronic infections of rodents worldwide . Invasion of human dwellings by infected rodents can result in human infections through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW .",
"Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW . The OW Lassa virus LASV , causative agent of Lassa fever LF , is the most significant OW mammarenaviral pathogen. LASV is estimated to infect several hundred thousand individuals annually in Western Africa, resulting in a high number of LF cases associated with high morbidity and lethality. Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever .",
"Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever . The worldwide-distributed OW mammarenavirus lymphocytic choriomeningitis virus LCMV is a neglected human pathogen of clinical significance especially in congenital infections . Moreover, LCMV poses a particular threat to immunocompromised individuals, as has been illustrated by fatal cases of LCMV infection associated with organ transplants . However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina.",
"However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina. Likewise, current anti-mammarenavirus therapy is limited to an offlabel use of the nucleoside analogue ribavirin that is only partially effective and can cause significant side effects . Development of effective anti-mammarenavirus drugs has been hampered by the lack of detailed understanding of virus/host-cell interactions required for mammarenavirus multiplication that could represent amenable targets for antiviral therapy. the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B .",
"the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B . To facilitate the identification of specific PPI between NP and host cell proteins, we used our mammarenavirus tri-segmented r3 platform to design an r3LCMV expressing a C-terminus Strep-tag version of enhanced green fluorescent protein r3LCMV/eGFP-Strep that we used as a negative control Fig 1A and 1B . We rescued rLCMV/Strep-NP and r3LCMV/eGFP-Strep and confirmed the expression of strep-tagged NP and eGFP in rLCMV/ Strep-NP-and r3LCMV/eGFP-Strep-infected cells, respectively Fig 1C . Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV.",
"Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV. However, both rLCMV/Strep-NP and r3LCMV/eGFP-Strep had WT-like growth kinetics and reached high titers. As with WT LCMV, infection with rLCMV/Strep-NP prevented production of bioactive IFN-I by cells in response to Sendai virus SeV infection as determined using an IFN bioassay based on protection against the cytopathic effect CPE induced by infection with vesicular stomatitis virus VSV Fig 1E . Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE.",
"Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE. We selected the human A549 cell line because lung epithelial cells are one of the initial cell targets of humans following inhalation of mammarenavirions. We infected A549 cells multiplicity of infection MOI = 0.1 with either rLCMV/Strep-NP or r3LCMV/eGFP-Strep Fig 2A . At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining.",
"At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining. We compared the pattern of stained protein bands detected between rLCMV/Strep-NP-and r3LCMV/eGFP-Strepinfected samples and confirmed the presence of Strep-NP and eGFP-Strep in pertinent samples Fig 2B . Protein complexes in the rest of eluates from the PD samples were concentrated by trichloroacetic acid TCA precipitation and subjected to trypsin digestion Fig 2A . Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 .",
"Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 . Filtering the data using these criteria resulted in identification of 139 candidate host-cell proteins as NP-interacting partners. Among 53 proteins found present in both NP-and eGFP-PD samples, 36 had spectral counts in the NP-PD sample that were less than two-fold higher than their corresponding spectral counts in the eGFP-PD sample Fig 2C and S1 Table . Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV.",
"Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV. The protein annotation through evolutionary relationship PANTHER protein family classification Biological Process of the NP-interacting protein candidates revealed that a large number of proteins were involved in metabolic and cellular processes Fig 2D . We also analyzed the molecular functions of the NP-interacting protein candidates according to the PAN-THER protein profile classification Fig 2E , which revealed diversified biochemical functions enriched for nucleic acid-binding proteins and chaperones. To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene .",
"To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene . OTPs are latest generation of siRNAs and offer significant advantages over previous generations. Off-target effects are primarily driven by antisense strand microRNA miR -like seed activity. In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy.",
"In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy. Numerous host-cell factors showed an anti-LCMV effect increased ZsG expression by kd of the genes , including microtubule-associated protein 1B MAP1B , dengue virus 2 DENV-2 , and chikungunya virus CHIKV . To begin assessing the potential biological implications of the identified NP-host cell protein interactions, we selected ATP1A1 and PHB given the availability of reagents, existing knowledge about their roles in cell physiology, and evidence of participation in multiplication of other viruses. We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers.",
"We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers. For this, we transfected A549 cells with siRNA targeting Expression of Strep-tagged proteins. A549 cells seeded 5.0 x 10 5 cells/well in a 6-well plate and cultured overnight were infected MOI = 0.1 with the indicated rLCMVs. At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins.",
"At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins. BHK-21 1.75 x 10 5 cells/well , A549 1.25 x 10 5 cells/well , or Vero E6 1.25 x 10 5 cells/well cells seeded in 24-well plates and cultured overnight were infected MOI = 0.01 with the indicated rLCMVs. At the indicated times pi, TCSs were collected and virus titers determined by IFFA. Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP.",
"Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP. A549 cells were infected MOI = 0.1 with the indicated rLCMV or mock-infected, and 36 h later infected with SeV MOI = 1 . At 24 h pi with SeV, TCS were collected and used, following virus inactivation by U.V., to treat Vero E6 cells for 16 h, followed by infection with rVSV MOI = 0.1 rVSV + or mockinfection rVSV - . At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production.",
"At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production. ATP1A1 or with NC siRNA 72 h prior to infection with rLCMV/ZsG. We found that siRNAmediated kd of ATP1A1 dramatically inhibited ZsGreen expression Fig 3Ci , which was associated with a significant reduction of infectious LCMV progeny Fig 3Cii . Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both.",
"Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both. Another consideration is that only a minor fraction of NP might be engaged in the interaction with a given host cell protein, and therefore, detection of these interactions would require highly sensitive methods such as LC-MS/MS. To overcome this problem we used confocal microscopy to examine the co-localization of NP with ATP1A1 and PHB in LCMV-infected cells. Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries .",
"Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries . The PHB inhibitor rocaglamide is a flavagline from an Aglaia tree used in traditional Chinese medicine that has potent anticancer activity . To examine whether pharmacological inhibition of ATP1A1 or PHB inhibited LCMV multiplication, we pretreated human A549 and HEK 293T , nonhuman primate Vero E6 , and rodent murine L929 and hamster BHK-21 cells with ouabain or rocaglamide and infected them with rLCMV/eGFP S1 Fig . Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition .",
"Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition . Likewise, we observed a dose-dependent rocaglamide inhibition of eGFP expression in all cell lines infected with rLCMV/eGFP S1B Fig . Consistent with these findings, production of infectious LCMV progeny was reduced by treatment with either ouabain or rocaglamide Fig 5A within a concentration range that had minimal impact on cell viability Fig 5B . To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability.",
"To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability. To gain insights about the mechanisms by which ouabain and rocaglamide exert their anti-LCMV activity, we examined effects of these compounds on distinct steps of the LCMV life cycle. First, we asked whether ouabain and rocaglamide affected cell entry of LCMV. We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection.",
"We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection. The timing of compound addition did not significantly change the number of eGFP-positive cells, indicating that neither ouabain nor rocaglamide inhibited cell entry of LCMV. The number of eGFP + cells in ouabain-treated cells was reduced at all time-of-addition points compared to vehicle dimethyl sulfoxide DMSO -treated cells, but was similar to that observed in ammonium chloride-treated cells. Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription.",
"Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription. To further investigate the effect of ouabain and rocaglamide on virus RNA synthesis, we infected A549 cells with a recombinant single-cycle infectious LCMV expressing eGFP rLCMVΔGPC/eGFP and treated cells with either ouabain or rocaglamide. Seventy-two hours later, we determined percent normalized eGFP expression in infected cells Fig 6B . Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis.",
"Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis. We also examined the effect of ouabain and rocaglamide on the late step of the arenavirus life cycle, Z-mediated budding. For this experiment, we transfected cells with Z-Strep-and Z-FLAG DYKDDDDK epitope -expressing plasmids from LCMV and LASV, respectively. At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide.",
"At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide. At 24 h, we determined by WB levels of Z protein in both whole cell lysates and associated with virus-like particles VLPs collected from TCS. Treatment with rocaglamide, but not with ouabain, caused a reduction in both LCMV and LASV Z budding efficiency Fig 6C and 6D . The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments.",
"The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments. The first experiment involved the use of a newly generated single-cycle infectious recombinant LCMV expressing the reporter gene ZsGreen scrLCMV/ZsG-P2A-NP to infect MOI = 0.1 A549 cells 1 st infection . These cells were subsequently transfected with a plasmid expressing LCMV GPC. After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F .",
"After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F . The second experiment involved infection MOI = 0.1 of cells with WT LCMV, and 48 h later we washed infected cells three times to remove the extracellular infectious progeny produced during the first 48 h of infection. Then, fresh media containing ouabain or DMSO vehicle control were added, and 24 h later we determined virus titers in TCS Fig 6G . Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections.",
"Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections. Since ouabain and rocaglamide inhibited different steps of the LCMV life cycle, we examined whether their use in combination results in a synergistic anti-LCMV effect. For this experiment, we infected A549 cells with rLCMV/eGFP and treated them with ouabain and rocaglamide using different concentrations and combinations. At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide.",
"At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide. We next asked whether the ATP1A1 and PHB host-cell factors contributed also to multiplication of viral hemorrhagic fever-causing LASV. We treated A549 cells with ouabain, bufalin, or rocaglamide and inoculated the treated cells with recombinant LASV expressing eGFP rLASV/eGFP . eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 .",
"eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 . We also tested the effect of compounds targeting ATP1A1 and PHB on multiplication of JUNV. Consistent with our results obtained with LCMV and LASV, ouabain, bufalin, and rocaglamide strongly suppressed JUNV multiplication S3 Fig . These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses.",
"These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses. Our approach using a recombinant LCMV expressing NP with an affinity tag facilitated defining the NP interactome in the context of LCMV infection. Recently, using an NP-specific monoclonal antibody mAb to precipitate NP and associated cellular protein partners in a mammarenavirus NP interactome, King et al. identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al.",
"identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al. Differences in both experimental conditions and analysis methodologies used to generate the LCMV NP interactome documented by King et al. and ours likely h post-transfection, cells were washed with fresh media to eliminate Z-mediated production of VLPs in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain or Roc-A at the indicated concentrations. VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D .",
"VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D . Budding efficiency for each sample was estimated by dividing the signal intensity of the Z protein associated with VLPs by that of Z detected in the cell lysate. Numbers on the bottom of panel C correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments.",
"Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments. F Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected MOI = 0.1 with scrLCMV/ZsG 1 st infection for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared.",
"At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. ZsGreen signal intensity was measured by a fluorescent plate reader. GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%.",
"GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%. Data represent means ± SD from three independent experiments. contributed to the observed differences between data sets. Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems.",
"Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems. We used results of the eGFP-Strep interactome, determined in r3LCMV/eGFP-Strep-infected cells, as a control to filter out non-specific NP interactions, which may have resulted in a higher degree of stringency than in the study by King et al for selection of NP-interacting candidates. The combined information provided by the NP interactome reported by King et al. and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction .",
"and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction . The anti-IFN activity of NP was mapped to its C-terminal part and linked to the 3'-5' exonuclease domain present with the NP C-terminal part . Inhibitor-B kinase ε IKKε was identified as an NP-binding protein using plasmid-mediated overexpression in transfected cells , and the NP-IKKε binding affinity correlated with NP's ability to inhibit IFN-I induction . We, as well as the work by King et al. , did not detect this NP-IKKε interaction.",
"We, as well as the work by King et al. , did not detect this NP-IKKε interaction. This discrepancy may have been caused by very low expression of IKKε in LCMV-infected cells, which prevented detection of IKKε by LC-MS/MS. Alternatively, NP-IKKε interaction could possibility be temporarily regulated and take place at early times pi, but could be mostly absent at 48 h pi, the time at which we prepared the cell lysates for our proteomics studies. Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit .",
"Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit . ATP1A1 represents one of four α subunits . Recent evidence has suggested that the Na + /K + -ATPase is involved in multiple cell signaling pathways that are independent of its ion-pumping function . Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase .",
"Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase . Thus, a low concentration of ouabain induces a conformational change in ATP1A1 that results in activation and release of proto-oncogene tyrosine protein kinase, Src, from ATP1A1, followed by activation of as yet unknown downstream signaling that inhibits, for instance, cell entry of murine hepatitis virus MHV . However, our results indicated that ouabain did not interfere with LCMV cell entry. In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV.",
"In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV. Moreover, cell entry of JUNV occurs also by clathrin-mediated endocytosis , a process affected by Src signaling. Ouabain has been clinically used in several European countries for the management of congestive heart failure, whereas bufalin has been tested in clinical trials for cancer treatments , and the CST digoxin has been FDA-approved since 1997 to treat heart failure and atrial fibrillation. Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry.",
"Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry. In contrast, PHB was reported to be a cell entry receptor for DENV-2 and CHIKV . On the other hand, PHB did not act as a virus cell entry receptor for HCV. Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV .",
"Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV . These findings indicate that PHB is involved in different steps of the life cycle of a variety of viruses, and thereby an attractive target for the development of broad-spectrum antiviral drugs. Rocaglate is a group of natural compounds, which includes rocaglamide, that inhibits protein synthesis by targeting the ATP-dependent DEAD-box RNA helicase eukaryotic initiation factor 4A eIF4A and exerts anti-tumor activity . The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication.",
"The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication. These proteins, which included MAP1B, might have anti-LCMV activity. MAP1B has been shown to bind to nonstructural proteins 1 NS1 and 2 NS2 of human respiratory syncytial virus HRSV . NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined .",
"NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined . Examining the role of NP-MAP1B interaction in modulating NP's ability to inhibit induction of type I IFN is of interest. We identified among the NP-interacting host cell proteins the RNA helicase Moloney leukemia virus 10 MOV10 , which has been reported to be an antiviral factor for FLUAV , retroviruses , and DENV-2 . We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions.",
"We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions. MOV10 was shown to enhance IRF-3-mediated IFN-I induction following SeV infection through a tank binding kinase 1 TBK1 -independent and IKKε-dependent manner. This finding was further supported by demonstrating MOV10-IKKε interaction by co-immunoprecipitation studies . We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined.",
"We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined. Several members of the mammalian chaperonin-containing T-complex CCT were identified as prominent hits in our NP interactome. The mammalian CCT is critical for folding of many proteins with important functions in diverse cellular processes , and may protect complex protein topologies within its central cavity during biosynthesis and folding . The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV .",
"The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV . However, the role of these CCT proteins in virus multiplication remains unknown and may involve functions other than acting as molecular chaperones. Previous studies documented the presence of several components of the of eIF4F, including 4A, within viral replication-transcription complexes RTC detected in cells infected with LCMV and TCRV . These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity.",
"These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity. In this work, we documented the generation of rLCMV/Strep-NP and its use to define the NP-interactome in infected cells. We presented evidence that ATP1A1 and PHB contribute to efficient multiplication of mammarenaviruses using genetics and pharmacological inhibition of the genes. Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins.",
"Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins. Future studies elucidating the roles of pro-and antiviral host-cell factors identified in this study in mammarenavirus multiplication will advance our understanding of the multiple functions of NP and uncover novel cellular targets for the development of antimammarenaviral drugs. In addition, by identifying proviral host-cell factors, drugs that are already approved can be repurposing as therapeutics to combat human pathogenic mammarenavirus infections. Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described .",
"Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described . Generation of rLCMV/NP D382A and SeV, strain Cantell, was described 30, 82, 83 . An rLCMV lacking GPC and expressing eGFP rLCMVΔGPC/eGFP was generated by reverse genetics using procedures previously described . rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B .",
"rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B . The rLCMV expressing eGFP rLCMV/eGFP was generated as described , and the rLCMV expressing ZsGreen rLCMV/ZsG instead of eGFP was generated by reverse genetics using similar procedures to generate rLCMV/eGFP. Generation of rLASV expressing eGFP rLASV/eGFP will be described elsewhere. A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG.",
"A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG. scrLCMV/ZsG-P2A-NP was rescued by reverse genetics using similar procedures to generate rLCMVΔGPC/eGFP . LCMV titers were determined by immunofocus forming assay IFFA as described . Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA .",
"Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA . VSV titers were determined by a plaque assay. Total cell lysates were prepared in PD lysis buffer + 250 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% TritonX-100, 10% glycerol, 1 mM of MgCl 2 , 1 μM of CaCl 2 , 1 μM of ZnCl 2 and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA .",
"Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA . To detect Strep-tagged proteins, membranes were reacted with mouse monoclonal antibodies to Strep QIAGEN, Germantown, MD , eGFP Takara Bio USA, Mountain View, CA , GP 2 We33/ 36 , ATP1A1 TehrmoFisher Scientific, Rockford, IL , PHB Abcam, Cambridge, MA or rabbit polyclonal antibodies to α-tubulin Cell Signaling Technologies, Danvers, MA or glyceraldehyde-3-phosphate dehydrogenase GAPDH, Millipore , respectively, followed by incubation with appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G IgG antibodies Jackson ImmunoResearch Laboratories, West Grove, PA . SuperSignal West Pico or Femto chemiluminescent substrate Thermo Fisher Scientific was used to elicit chemiluminescent signals that were visualized using ImageQuant LAS 4000 Imager GE Healthcare Bio-Sciences, Pittsburgh, PA . Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1.",
"Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1. At 48 h pi, cells were washed three times with ice-cold PBS, scraped into fresh ice-cold PBS, and centrifuged at 400 x g at 4˚C for 10 min. Supernatant was removed, and cells were lysed with 12 ml of PD lysis buffer + supplemented with halt protease and phosphatase inhibitor cocktail Thermo Fisher Scientific and 5 μg/ml of deoxyribonuclease I Worthington Biochemical Corporation, Lakewood, NJ . Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C.",
"Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C. After 2 h of incubation, the resin was washed three times with PD lysis buffer + and once with PD lysis buffer without TritonX-100 PD lysis buffer - . After the centrifugation at 1,600 x g and 4˚C for 5 min, the last wash buffer was removed, and protein complexes associated with the resin were eluted into 2 ml of PD lysis buffer - containing 2.5 mM of desthiobiotin. The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn.",
"The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn. A MudPIT microcolumn was prepared by first creating a Kasil frit at one end of an un-deactivated 250-μm outside diameter OD capillary tube interior diameter of 360 μm Agilent Technologies, Inc., Santa Clara, CA . The Kasil frit was prepared by briefly dipping a 20-30-cm capillary tube in 300 μl of Kasil 1624 potassium silicate well-mixed solution PQ Corporation, Malvern, PA and 100 μl of formamide, curing at 100˚C for 4 h, and cutting the frit to a length of %2 mm. Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading.",
"Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading. MudPIT analysis. An analytical reversed-phase liquid chromatography column was generated by pulling a 100-μm interior diameter ID of 360 μm OD capillary tube Polymicro Technologies, Phoenix, AZ to 5-μm ID tip. Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid .",
"Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid . MudPIT and analytical columns were assembled using a zero-dead volume union Upchurch Scientific, Oak Harbor, WA . LC-MS/MS analysis was performed with an Agilent high-pressure LC pump Agilent and linear quadrupole ion dual cell trap Orbitrap Velos Thermo using an in-house built electrospray stage. Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient.",
"Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient. Data analysis. Protein and peptide identification were performed with Integrated Proteomics Pipeline-IP2 Integrated Proteomics Applications, San Diego, CA. integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in.",
"integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in. Spectrum raw files were extracted into ms2 files from raw files using open source RawExtract 1.9.9 Scripps Research Institute, La Jolla, CA; and the tandem mass spectra were searched against a human protein database UniprotKB . To accurately estimate peptide probabilities and false discovery rates, we used a decoy database containing the reversed sequences of all the proteins appended to the target database. Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system.",
"Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system. The search space included half and fully tryptic peptide candidates that fell within the mass tolerance window with no miscleavage constraint. Carbamidomethylation +57.02146 Da of cysteine was considered as a static modification. siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG.",
"siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG. siRNA target host-cell proteins were selected based on availability of validated siRNA sequences. The siRNAs we used to examine the effects on LCMV multiplication of knockdown expression of NP-interacting host cell protein candidate hits corresponded to the Genome-wide ON TAR-GET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene; Dharmacon, Lafayette, CO . A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min.",
"A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. The total protein concentration of clarified cell lysate was measured by Pierce BCA Protein Assay Kit Thermo Fisher Scientific . The same amount of protein from each sample was subjected to SDS-PAGE, and the protein expression of siRNA-targeted genes was analyzed by western blots. Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI .",
"Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI . Green fluorescence eGFP or ZsGreen and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, Bio-Tek, Winooski, VT . Mock-and virus-infected cells were fixed with 4% PFA. After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488.",
"After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488. In some samples, primary antibody against ATP1A1 or PHB was omitted to determine background fluorescence. To visualize nuclei, DAPI Fluoromount-G SouthernBiotech, Birmingham, AL was used to mount coverslips on a slide glass. Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss .",
"Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss . Eight green NP-positive cells were marked and every pixel in the marked area was plotted in the scatter diagram based on its intensity level from each channel. Thresholds for green and red channels were determined using mock-infected cells stained with VL-4-AF488 anti-NP and anti-mouse IgG antibody conjugated with Alexa Fluor 568, without using anti-ATP1A1 or -PHB antibodies. Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels.",
"Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels. This calculation was repeated for eight individual cells. To assess the specificity of co-localization, we determined weighted CC by taking into consideration the brightness of each channel signal. Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software.",
"Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software. A549 or Vero E6 cells seeded 2.0 x 10 4 cells/well in a 96-well plate and cultured overnight were treated with 3-fold serial compound dilutions at 37˚C and 5% CO 2 for 2 h, followed by infection with rLCMV/eGFP MOI = 0.01 . Compounds were present to study endpoint. At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%.",
"At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%. The IC 50 concentrations were determined using GraphPad Prism. A549 or Vero E6 cells seeded in a 96-well plate 2.0 x 10 4 cells/well and cultured overnight were treated with 3-fold serial compound dilutions and cultured at 37˚C and 5% CO 2 for 48 h. Then, CellTiter 96 AQ ueous one solution reagent Promega, Madison, WI was added. Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%.",
"Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%. The CC 50 concentrations were determined using GraphPad Prism. A plasmid expressing C-terminus Strep-tagged Z protein pC-LCMV-Z-Strep was generated using similar procedure to generate a plasmid expressing C-terminus FLAG-tagged LASV Z protein pC-LASV-Z-FLAG , and the budding assay was performed as previously described . Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium.",
"Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium. After the removal of the last wash medium, cells were cultured in fresh medium containing ouabain 30 or 40 nM or rocaglamide 50 or 100 nM or equivalent concentration of DMSO, and 24 h later, virion-like particle VLP -containing TCS and cells were collected. Total cell lysate was prepared by lysing the cells with lysis buffer 1% NP-40, 50 mM of Tris-HCl pH 8.0 , 62.5 mM NaCl, 0.4% sodium deoxycholate . After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots.",
"After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots. A549 cells infected with rLCMV/eGFP were harvested using Accutase cell detachment solution Innovative Cell Technologies, San Diego, CA and fixed with 4% PFA in PBS. eGFP expression was examined by flow cytometry using a BD LSR II Becton Dickson , and data were analyzed with FlowJo Tree Star, Inc., Ashland, OR . 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO .",
"293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO . At 48 h pi, TCS was collected and used to infect fresh monolayers of BHK-21 cells seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . A549 cells seeded 2 x 10 5 cells/well in a 96-well plate and cultured overnight were treated with combinations of different concentrations of ouabain and rocaglamide for 2 h and then infected MOI = 0.01 with rLCMV/eGFP. Compounds were present in the culture medium throughout the experiment. At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . eGFP readouts were normalized by DAPI readouts, and normalized data were used to analyze synergistic effect of the two compounds by the MacSynergy II program . Data were analyzed for p values by a two-tailed unpaired t test using GraphPad Prism software."
] | 1,687 | 5,152 |
Why was the human A549 cell line chosen for this study? | lung epithelial cells are one of the initial cell targets of humans following inhalation of mammarenavirions | [
"Several mammalian arenaviruses mammarenaviruses cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus LCMV , is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors.",
"Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.",
"We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. Text: Introduction Mammarenaviruses Arenaviridae: Mammarenavirus cause chronic infections of rodents worldwide . Invasion of human dwellings by infected rodents can result in human infections through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW .",
"Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW . The OW Lassa virus LASV , causative agent of Lassa fever LF , is the most significant OW mammarenaviral pathogen. LASV is estimated to infect several hundred thousand individuals annually in Western Africa, resulting in a high number of LF cases associated with high morbidity and lethality. Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever .",
"Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever . The worldwide-distributed OW mammarenavirus lymphocytic choriomeningitis virus LCMV is a neglected human pathogen of clinical significance especially in congenital infections . Moreover, LCMV poses a particular threat to immunocompromised individuals, as has been illustrated by fatal cases of LCMV infection associated with organ transplants . However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina.",
"However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina. Likewise, current anti-mammarenavirus therapy is limited to an offlabel use of the nucleoside analogue ribavirin that is only partially effective and can cause significant side effects . Development of effective anti-mammarenavirus drugs has been hampered by the lack of detailed understanding of virus/host-cell interactions required for mammarenavirus multiplication that could represent amenable targets for antiviral therapy. the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B .",
"the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B . To facilitate the identification of specific PPI between NP and host cell proteins, we used our mammarenavirus tri-segmented r3 platform to design an r3LCMV expressing a C-terminus Strep-tag version of enhanced green fluorescent protein r3LCMV/eGFP-Strep that we used as a negative control Fig 1A and 1B . We rescued rLCMV/Strep-NP and r3LCMV/eGFP-Strep and confirmed the expression of strep-tagged NP and eGFP in rLCMV/ Strep-NP-and r3LCMV/eGFP-Strep-infected cells, respectively Fig 1C . Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV.",
"Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV. However, both rLCMV/Strep-NP and r3LCMV/eGFP-Strep had WT-like growth kinetics and reached high titers. As with WT LCMV, infection with rLCMV/Strep-NP prevented production of bioactive IFN-I by cells in response to Sendai virus SeV infection as determined using an IFN bioassay based on protection against the cytopathic effect CPE induced by infection with vesicular stomatitis virus VSV Fig 1E . Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE.",
"Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE. We selected the human A549 cell line because lung epithelial cells are one of the initial cell targets of humans following inhalation of mammarenavirions. We infected A549 cells multiplicity of infection MOI = 0.1 with either rLCMV/Strep-NP or r3LCMV/eGFP-Strep Fig 2A . At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining.",
"At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining. We compared the pattern of stained protein bands detected between rLCMV/Strep-NP-and r3LCMV/eGFP-Strepinfected samples and confirmed the presence of Strep-NP and eGFP-Strep in pertinent samples Fig 2B . Protein complexes in the rest of eluates from the PD samples were concentrated by trichloroacetic acid TCA precipitation and subjected to trypsin digestion Fig 2A . Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 .",
"Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 . Filtering the data using these criteria resulted in identification of 139 candidate host-cell proteins as NP-interacting partners. Among 53 proteins found present in both NP-and eGFP-PD samples, 36 had spectral counts in the NP-PD sample that were less than two-fold higher than their corresponding spectral counts in the eGFP-PD sample Fig 2C and S1 Table . Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV.",
"Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV. The protein annotation through evolutionary relationship PANTHER protein family classification Biological Process of the NP-interacting protein candidates revealed that a large number of proteins were involved in metabolic and cellular processes Fig 2D . We also analyzed the molecular functions of the NP-interacting protein candidates according to the PAN-THER protein profile classification Fig 2E , which revealed diversified biochemical functions enriched for nucleic acid-binding proteins and chaperones. To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene .",
"To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene . OTPs are latest generation of siRNAs and offer significant advantages over previous generations. Off-target effects are primarily driven by antisense strand microRNA miR -like seed activity. In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy.",
"In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy. Numerous host-cell factors showed an anti-LCMV effect increased ZsG expression by kd of the genes , including microtubule-associated protein 1B MAP1B , dengue virus 2 DENV-2 , and chikungunya virus CHIKV . To begin assessing the potential biological implications of the identified NP-host cell protein interactions, we selected ATP1A1 and PHB given the availability of reagents, existing knowledge about their roles in cell physiology, and evidence of participation in multiplication of other viruses. We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers.",
"We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers. For this, we transfected A549 cells with siRNA targeting Expression of Strep-tagged proteins. A549 cells seeded 5.0 x 10 5 cells/well in a 6-well plate and cultured overnight were infected MOI = 0.1 with the indicated rLCMVs. At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins.",
"At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins. BHK-21 1.75 x 10 5 cells/well , A549 1.25 x 10 5 cells/well , or Vero E6 1.25 x 10 5 cells/well cells seeded in 24-well plates and cultured overnight were infected MOI = 0.01 with the indicated rLCMVs. At the indicated times pi, TCSs were collected and virus titers determined by IFFA. Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP.",
"Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP. A549 cells were infected MOI = 0.1 with the indicated rLCMV or mock-infected, and 36 h later infected with SeV MOI = 1 . At 24 h pi with SeV, TCS were collected and used, following virus inactivation by U.V., to treat Vero E6 cells for 16 h, followed by infection with rVSV MOI = 0.1 rVSV + or mockinfection rVSV - . At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production.",
"At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production. ATP1A1 or with NC siRNA 72 h prior to infection with rLCMV/ZsG. We found that siRNAmediated kd of ATP1A1 dramatically inhibited ZsGreen expression Fig 3Ci , which was associated with a significant reduction of infectious LCMV progeny Fig 3Cii . Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both.",
"Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both. Another consideration is that only a minor fraction of NP might be engaged in the interaction with a given host cell protein, and therefore, detection of these interactions would require highly sensitive methods such as LC-MS/MS. To overcome this problem we used confocal microscopy to examine the co-localization of NP with ATP1A1 and PHB in LCMV-infected cells. Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries .",
"Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries . The PHB inhibitor rocaglamide is a flavagline from an Aglaia tree used in traditional Chinese medicine that has potent anticancer activity . To examine whether pharmacological inhibition of ATP1A1 or PHB inhibited LCMV multiplication, we pretreated human A549 and HEK 293T , nonhuman primate Vero E6 , and rodent murine L929 and hamster BHK-21 cells with ouabain or rocaglamide and infected them with rLCMV/eGFP S1 Fig . Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition .",
"Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition . Likewise, we observed a dose-dependent rocaglamide inhibition of eGFP expression in all cell lines infected with rLCMV/eGFP S1B Fig . Consistent with these findings, production of infectious LCMV progeny was reduced by treatment with either ouabain or rocaglamide Fig 5A within a concentration range that had minimal impact on cell viability Fig 5B . To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability.",
"To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability. To gain insights about the mechanisms by which ouabain and rocaglamide exert their anti-LCMV activity, we examined effects of these compounds on distinct steps of the LCMV life cycle. First, we asked whether ouabain and rocaglamide affected cell entry of LCMV. We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection.",
"We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection. The timing of compound addition did not significantly change the number of eGFP-positive cells, indicating that neither ouabain nor rocaglamide inhibited cell entry of LCMV. The number of eGFP + cells in ouabain-treated cells was reduced at all time-of-addition points compared to vehicle dimethyl sulfoxide DMSO -treated cells, but was similar to that observed in ammonium chloride-treated cells. Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription.",
"Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription. To further investigate the effect of ouabain and rocaglamide on virus RNA synthesis, we infected A549 cells with a recombinant single-cycle infectious LCMV expressing eGFP rLCMVΔGPC/eGFP and treated cells with either ouabain or rocaglamide. Seventy-two hours later, we determined percent normalized eGFP expression in infected cells Fig 6B . Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis.",
"Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis. We also examined the effect of ouabain and rocaglamide on the late step of the arenavirus life cycle, Z-mediated budding. For this experiment, we transfected cells with Z-Strep-and Z-FLAG DYKDDDDK epitope -expressing plasmids from LCMV and LASV, respectively. At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide.",
"At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide. At 24 h, we determined by WB levels of Z protein in both whole cell lysates and associated with virus-like particles VLPs collected from TCS. Treatment with rocaglamide, but not with ouabain, caused a reduction in both LCMV and LASV Z budding efficiency Fig 6C and 6D . The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments.",
"The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments. The first experiment involved the use of a newly generated single-cycle infectious recombinant LCMV expressing the reporter gene ZsGreen scrLCMV/ZsG-P2A-NP to infect MOI = 0.1 A549 cells 1 st infection . These cells were subsequently transfected with a plasmid expressing LCMV GPC. After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F .",
"After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F . The second experiment involved infection MOI = 0.1 of cells with WT LCMV, and 48 h later we washed infected cells three times to remove the extracellular infectious progeny produced during the first 48 h of infection. Then, fresh media containing ouabain or DMSO vehicle control were added, and 24 h later we determined virus titers in TCS Fig 6G . Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections.",
"Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections. Since ouabain and rocaglamide inhibited different steps of the LCMV life cycle, we examined whether their use in combination results in a synergistic anti-LCMV effect. For this experiment, we infected A549 cells with rLCMV/eGFP and treated them with ouabain and rocaglamide using different concentrations and combinations. At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide.",
"At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide. We next asked whether the ATP1A1 and PHB host-cell factors contributed also to multiplication of viral hemorrhagic fever-causing LASV. We treated A549 cells with ouabain, bufalin, or rocaglamide and inoculated the treated cells with recombinant LASV expressing eGFP rLASV/eGFP . eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 .",
"eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 . We also tested the effect of compounds targeting ATP1A1 and PHB on multiplication of JUNV. Consistent with our results obtained with LCMV and LASV, ouabain, bufalin, and rocaglamide strongly suppressed JUNV multiplication S3 Fig . These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses.",
"These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses. Our approach using a recombinant LCMV expressing NP with an affinity tag facilitated defining the NP interactome in the context of LCMV infection. Recently, using an NP-specific monoclonal antibody mAb to precipitate NP and associated cellular protein partners in a mammarenavirus NP interactome, King et al. identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al.",
"identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al. Differences in both experimental conditions and analysis methodologies used to generate the LCMV NP interactome documented by King et al. and ours likely h post-transfection, cells were washed with fresh media to eliminate Z-mediated production of VLPs in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain or Roc-A at the indicated concentrations. VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D .",
"VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D . Budding efficiency for each sample was estimated by dividing the signal intensity of the Z protein associated with VLPs by that of Z detected in the cell lysate. Numbers on the bottom of panel C correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments.",
"Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments. F Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected MOI = 0.1 with scrLCMV/ZsG 1 st infection for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared.",
"At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. ZsGreen signal intensity was measured by a fluorescent plate reader. GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%.",
"GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%. Data represent means ± SD from three independent experiments. contributed to the observed differences between data sets. Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems.",
"Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems. We used results of the eGFP-Strep interactome, determined in r3LCMV/eGFP-Strep-infected cells, as a control to filter out non-specific NP interactions, which may have resulted in a higher degree of stringency than in the study by King et al for selection of NP-interacting candidates. The combined information provided by the NP interactome reported by King et al. and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction .",
"and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction . The anti-IFN activity of NP was mapped to its C-terminal part and linked to the 3'-5' exonuclease domain present with the NP C-terminal part . Inhibitor-B kinase ε IKKε was identified as an NP-binding protein using plasmid-mediated overexpression in transfected cells , and the NP-IKKε binding affinity correlated with NP's ability to inhibit IFN-I induction . We, as well as the work by King et al. , did not detect this NP-IKKε interaction.",
"We, as well as the work by King et al. , did not detect this NP-IKKε interaction. This discrepancy may have been caused by very low expression of IKKε in LCMV-infected cells, which prevented detection of IKKε by LC-MS/MS. Alternatively, NP-IKKε interaction could possibility be temporarily regulated and take place at early times pi, but could be mostly absent at 48 h pi, the time at which we prepared the cell lysates for our proteomics studies. Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit .",
"Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit . ATP1A1 represents one of four α subunits . Recent evidence has suggested that the Na + /K + -ATPase is involved in multiple cell signaling pathways that are independent of its ion-pumping function . Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase .",
"Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase . Thus, a low concentration of ouabain induces a conformational change in ATP1A1 that results in activation and release of proto-oncogene tyrosine protein kinase, Src, from ATP1A1, followed by activation of as yet unknown downstream signaling that inhibits, for instance, cell entry of murine hepatitis virus MHV . However, our results indicated that ouabain did not interfere with LCMV cell entry. In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV.",
"In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV. Moreover, cell entry of JUNV occurs also by clathrin-mediated endocytosis , a process affected by Src signaling. Ouabain has been clinically used in several European countries for the management of congestive heart failure, whereas bufalin has been tested in clinical trials for cancer treatments , and the CST digoxin has been FDA-approved since 1997 to treat heart failure and atrial fibrillation. Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry.",
"Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry. In contrast, PHB was reported to be a cell entry receptor for DENV-2 and CHIKV . On the other hand, PHB did not act as a virus cell entry receptor for HCV. Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV .",
"Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV . These findings indicate that PHB is involved in different steps of the life cycle of a variety of viruses, and thereby an attractive target for the development of broad-spectrum antiviral drugs. Rocaglate is a group of natural compounds, which includes rocaglamide, that inhibits protein synthesis by targeting the ATP-dependent DEAD-box RNA helicase eukaryotic initiation factor 4A eIF4A and exerts anti-tumor activity . The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication.",
"The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication. These proteins, which included MAP1B, might have anti-LCMV activity. MAP1B has been shown to bind to nonstructural proteins 1 NS1 and 2 NS2 of human respiratory syncytial virus HRSV . NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined .",
"NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined . Examining the role of NP-MAP1B interaction in modulating NP's ability to inhibit induction of type I IFN is of interest. We identified among the NP-interacting host cell proteins the RNA helicase Moloney leukemia virus 10 MOV10 , which has been reported to be an antiviral factor for FLUAV , retroviruses , and DENV-2 . We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions.",
"We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions. MOV10 was shown to enhance IRF-3-mediated IFN-I induction following SeV infection through a tank binding kinase 1 TBK1 -independent and IKKε-dependent manner. This finding was further supported by demonstrating MOV10-IKKε interaction by co-immunoprecipitation studies . We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined.",
"We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined. Several members of the mammalian chaperonin-containing T-complex CCT were identified as prominent hits in our NP interactome. The mammalian CCT is critical for folding of many proteins with important functions in diverse cellular processes , and may protect complex protein topologies within its central cavity during biosynthesis and folding . The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV .",
"The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV . However, the role of these CCT proteins in virus multiplication remains unknown and may involve functions other than acting as molecular chaperones. Previous studies documented the presence of several components of the of eIF4F, including 4A, within viral replication-transcription complexes RTC detected in cells infected with LCMV and TCRV . These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity.",
"These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity. In this work, we documented the generation of rLCMV/Strep-NP and its use to define the NP-interactome in infected cells. We presented evidence that ATP1A1 and PHB contribute to efficient multiplication of mammarenaviruses using genetics and pharmacological inhibition of the genes. Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins.",
"Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins. Future studies elucidating the roles of pro-and antiviral host-cell factors identified in this study in mammarenavirus multiplication will advance our understanding of the multiple functions of NP and uncover novel cellular targets for the development of antimammarenaviral drugs. In addition, by identifying proviral host-cell factors, drugs that are already approved can be repurposing as therapeutics to combat human pathogenic mammarenavirus infections. Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described .",
"Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described . Generation of rLCMV/NP D382A and SeV, strain Cantell, was described 30, 82, 83 . An rLCMV lacking GPC and expressing eGFP rLCMVΔGPC/eGFP was generated by reverse genetics using procedures previously described . rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B .",
"rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B . The rLCMV expressing eGFP rLCMV/eGFP was generated as described , and the rLCMV expressing ZsGreen rLCMV/ZsG instead of eGFP was generated by reverse genetics using similar procedures to generate rLCMV/eGFP. Generation of rLASV expressing eGFP rLASV/eGFP will be described elsewhere. A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG.",
"A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG. scrLCMV/ZsG-P2A-NP was rescued by reverse genetics using similar procedures to generate rLCMVΔGPC/eGFP . LCMV titers were determined by immunofocus forming assay IFFA as described . Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA .",
"Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA . VSV titers were determined by a plaque assay. Total cell lysates were prepared in PD lysis buffer + 250 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% TritonX-100, 10% glycerol, 1 mM of MgCl 2 , 1 μM of CaCl 2 , 1 μM of ZnCl 2 and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA .",
"Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA . To detect Strep-tagged proteins, membranes were reacted with mouse monoclonal antibodies to Strep QIAGEN, Germantown, MD , eGFP Takara Bio USA, Mountain View, CA , GP 2 We33/ 36 , ATP1A1 TehrmoFisher Scientific, Rockford, IL , PHB Abcam, Cambridge, MA or rabbit polyclonal antibodies to α-tubulin Cell Signaling Technologies, Danvers, MA or glyceraldehyde-3-phosphate dehydrogenase GAPDH, Millipore , respectively, followed by incubation with appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G IgG antibodies Jackson ImmunoResearch Laboratories, West Grove, PA . SuperSignal West Pico or Femto chemiluminescent substrate Thermo Fisher Scientific was used to elicit chemiluminescent signals that were visualized using ImageQuant LAS 4000 Imager GE Healthcare Bio-Sciences, Pittsburgh, PA . Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1.",
"Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1. At 48 h pi, cells were washed three times with ice-cold PBS, scraped into fresh ice-cold PBS, and centrifuged at 400 x g at 4˚C for 10 min. Supernatant was removed, and cells were lysed with 12 ml of PD lysis buffer + supplemented with halt protease and phosphatase inhibitor cocktail Thermo Fisher Scientific and 5 μg/ml of deoxyribonuclease I Worthington Biochemical Corporation, Lakewood, NJ . Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C.",
"Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C. After 2 h of incubation, the resin was washed three times with PD lysis buffer + and once with PD lysis buffer without TritonX-100 PD lysis buffer - . After the centrifugation at 1,600 x g and 4˚C for 5 min, the last wash buffer was removed, and protein complexes associated with the resin were eluted into 2 ml of PD lysis buffer - containing 2.5 mM of desthiobiotin. The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn.",
"The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn. A MudPIT microcolumn was prepared by first creating a Kasil frit at one end of an un-deactivated 250-μm outside diameter OD capillary tube interior diameter of 360 μm Agilent Technologies, Inc., Santa Clara, CA . The Kasil frit was prepared by briefly dipping a 20-30-cm capillary tube in 300 μl of Kasil 1624 potassium silicate well-mixed solution PQ Corporation, Malvern, PA and 100 μl of formamide, curing at 100˚C for 4 h, and cutting the frit to a length of %2 mm. Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading.",
"Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading. MudPIT analysis. An analytical reversed-phase liquid chromatography column was generated by pulling a 100-μm interior diameter ID of 360 μm OD capillary tube Polymicro Technologies, Phoenix, AZ to 5-μm ID tip. Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid .",
"Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid . MudPIT and analytical columns were assembled using a zero-dead volume union Upchurch Scientific, Oak Harbor, WA . LC-MS/MS analysis was performed with an Agilent high-pressure LC pump Agilent and linear quadrupole ion dual cell trap Orbitrap Velos Thermo using an in-house built electrospray stage. Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient.",
"Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient. Data analysis. Protein and peptide identification were performed with Integrated Proteomics Pipeline-IP2 Integrated Proteomics Applications, San Diego, CA. integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in.",
"integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in. Spectrum raw files were extracted into ms2 files from raw files using open source RawExtract 1.9.9 Scripps Research Institute, La Jolla, CA; and the tandem mass spectra were searched against a human protein database UniprotKB . To accurately estimate peptide probabilities and false discovery rates, we used a decoy database containing the reversed sequences of all the proteins appended to the target database. Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system.",
"Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system. The search space included half and fully tryptic peptide candidates that fell within the mass tolerance window with no miscleavage constraint. Carbamidomethylation +57.02146 Da of cysteine was considered as a static modification. siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG.",
"siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG. siRNA target host-cell proteins were selected based on availability of validated siRNA sequences. The siRNAs we used to examine the effects on LCMV multiplication of knockdown expression of NP-interacting host cell protein candidate hits corresponded to the Genome-wide ON TAR-GET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene; Dharmacon, Lafayette, CO . A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min.",
"A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. The total protein concentration of clarified cell lysate was measured by Pierce BCA Protein Assay Kit Thermo Fisher Scientific . The same amount of protein from each sample was subjected to SDS-PAGE, and the protein expression of siRNA-targeted genes was analyzed by western blots. Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI .",
"Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI . Green fluorescence eGFP or ZsGreen and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, Bio-Tek, Winooski, VT . Mock-and virus-infected cells were fixed with 4% PFA. After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488.",
"After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488. In some samples, primary antibody against ATP1A1 or PHB was omitted to determine background fluorescence. To visualize nuclei, DAPI Fluoromount-G SouthernBiotech, Birmingham, AL was used to mount coverslips on a slide glass. Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss .",
"Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss . Eight green NP-positive cells were marked and every pixel in the marked area was plotted in the scatter diagram based on its intensity level from each channel. Thresholds for green and red channels were determined using mock-infected cells stained with VL-4-AF488 anti-NP and anti-mouse IgG antibody conjugated with Alexa Fluor 568, without using anti-ATP1A1 or -PHB antibodies. Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels.",
"Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels. This calculation was repeated for eight individual cells. To assess the specificity of co-localization, we determined weighted CC by taking into consideration the brightness of each channel signal. Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software.",
"Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software. A549 or Vero E6 cells seeded 2.0 x 10 4 cells/well in a 96-well plate and cultured overnight were treated with 3-fold serial compound dilutions at 37˚C and 5% CO 2 for 2 h, followed by infection with rLCMV/eGFP MOI = 0.01 . Compounds were present to study endpoint. At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%.",
"At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%. The IC 50 concentrations were determined using GraphPad Prism. A549 or Vero E6 cells seeded in a 96-well plate 2.0 x 10 4 cells/well and cultured overnight were treated with 3-fold serial compound dilutions and cultured at 37˚C and 5% CO 2 for 48 h. Then, CellTiter 96 AQ ueous one solution reagent Promega, Madison, WI was added. Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%.",
"Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%. The CC 50 concentrations were determined using GraphPad Prism. A plasmid expressing C-terminus Strep-tagged Z protein pC-LCMV-Z-Strep was generated using similar procedure to generate a plasmid expressing C-terminus FLAG-tagged LASV Z protein pC-LASV-Z-FLAG , and the budding assay was performed as previously described . Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium.",
"Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium. After the removal of the last wash medium, cells were cultured in fresh medium containing ouabain 30 or 40 nM or rocaglamide 50 or 100 nM or equivalent concentration of DMSO, and 24 h later, virion-like particle VLP -containing TCS and cells were collected. Total cell lysate was prepared by lysing the cells with lysis buffer 1% NP-40, 50 mM of Tris-HCl pH 8.0 , 62.5 mM NaCl, 0.4% sodium deoxycholate . After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots.",
"After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots. A549 cells infected with rLCMV/eGFP were harvested using Accutase cell detachment solution Innovative Cell Technologies, San Diego, CA and fixed with 4% PFA in PBS. eGFP expression was examined by flow cytometry using a BD LSR II Becton Dickson , and data were analyzed with FlowJo Tree Star, Inc., Ashland, OR . 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO .",
"293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO . At 48 h pi, TCS was collected and used to infect fresh monolayers of BHK-21 cells seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . A549 cells seeded 2 x 10 5 cells/well in a 96-well plate and cultured overnight were treated with combinations of different concentrations of ouabain and rocaglamide for 2 h and then infected MOI = 0.01 with rLCMV/eGFP. Compounds were present in the culture medium throughout the experiment. At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . eGFP readouts were normalized by DAPI readouts, and normalized data were used to analyze synergistic effect of the two compounds by the MacSynergy II program . Data were analyzed for p values by a two-tailed unpaired t test using GraphPad Prism software."
] | 1,687 | 5,153 |
For how long were the cells infected before analysis? | 48 h | [
"Several mammalian arenaviruses mammarenaviruses cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus LCMV , is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors.",
"Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.",
"We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. Text: Introduction Mammarenaviruses Arenaviridae: Mammarenavirus cause chronic infections of rodents worldwide . Invasion of human dwellings by infected rodents can result in human infections through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW .",
"Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW . The OW Lassa virus LASV , causative agent of Lassa fever LF , is the most significant OW mammarenaviral pathogen. LASV is estimated to infect several hundred thousand individuals annually in Western Africa, resulting in a high number of LF cases associated with high morbidity and lethality. Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever .",
"Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever . The worldwide-distributed OW mammarenavirus lymphocytic choriomeningitis virus LCMV is a neglected human pathogen of clinical significance especially in congenital infections . Moreover, LCMV poses a particular threat to immunocompromised individuals, as has been illustrated by fatal cases of LCMV infection associated with organ transplants . However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina.",
"However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina. Likewise, current anti-mammarenavirus therapy is limited to an offlabel use of the nucleoside analogue ribavirin that is only partially effective and can cause significant side effects . Development of effective anti-mammarenavirus drugs has been hampered by the lack of detailed understanding of virus/host-cell interactions required for mammarenavirus multiplication that could represent amenable targets for antiviral therapy. the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B .",
"the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B . To facilitate the identification of specific PPI between NP and host cell proteins, we used our mammarenavirus tri-segmented r3 platform to design an r3LCMV expressing a C-terminus Strep-tag version of enhanced green fluorescent protein r3LCMV/eGFP-Strep that we used as a negative control Fig 1A and 1B . We rescued rLCMV/Strep-NP and r3LCMV/eGFP-Strep and confirmed the expression of strep-tagged NP and eGFP in rLCMV/ Strep-NP-and r3LCMV/eGFP-Strep-infected cells, respectively Fig 1C . Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV.",
"Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV. However, both rLCMV/Strep-NP and r3LCMV/eGFP-Strep had WT-like growth kinetics and reached high titers. As with WT LCMV, infection with rLCMV/Strep-NP prevented production of bioactive IFN-I by cells in response to Sendai virus SeV infection as determined using an IFN bioassay based on protection against the cytopathic effect CPE induced by infection with vesicular stomatitis virus VSV Fig 1E . Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE.",
"Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE. We selected the human A549 cell line because lung epithelial cells are one of the initial cell targets of humans following inhalation of mammarenavirions. We infected A549 cells multiplicity of infection MOI = 0.1 with either rLCMV/Strep-NP or r3LCMV/eGFP-Strep Fig 2A . At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining.",
"At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining. We compared the pattern of stained protein bands detected between rLCMV/Strep-NP-and r3LCMV/eGFP-Strepinfected samples and confirmed the presence of Strep-NP and eGFP-Strep in pertinent samples Fig 2B . Protein complexes in the rest of eluates from the PD samples were concentrated by trichloroacetic acid TCA precipitation and subjected to trypsin digestion Fig 2A . Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 .",
"Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 . Filtering the data using these criteria resulted in identification of 139 candidate host-cell proteins as NP-interacting partners. Among 53 proteins found present in both NP-and eGFP-PD samples, 36 had spectral counts in the NP-PD sample that were less than two-fold higher than their corresponding spectral counts in the eGFP-PD sample Fig 2C and S1 Table . Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV.",
"Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV. The protein annotation through evolutionary relationship PANTHER protein family classification Biological Process of the NP-interacting protein candidates revealed that a large number of proteins were involved in metabolic and cellular processes Fig 2D . We also analyzed the molecular functions of the NP-interacting protein candidates according to the PAN-THER protein profile classification Fig 2E , which revealed diversified biochemical functions enriched for nucleic acid-binding proteins and chaperones. To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene .",
"To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene . OTPs are latest generation of siRNAs and offer significant advantages over previous generations. Off-target effects are primarily driven by antisense strand microRNA miR -like seed activity. In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy.",
"In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy. Numerous host-cell factors showed an anti-LCMV effect increased ZsG expression by kd of the genes , including microtubule-associated protein 1B MAP1B , dengue virus 2 DENV-2 , and chikungunya virus CHIKV . To begin assessing the potential biological implications of the identified NP-host cell protein interactions, we selected ATP1A1 and PHB given the availability of reagents, existing knowledge about their roles in cell physiology, and evidence of participation in multiplication of other viruses. We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers.",
"We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers. For this, we transfected A549 cells with siRNA targeting Expression of Strep-tagged proteins. A549 cells seeded 5.0 x 10 5 cells/well in a 6-well plate and cultured overnight were infected MOI = 0.1 with the indicated rLCMVs. At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins.",
"At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins. BHK-21 1.75 x 10 5 cells/well , A549 1.25 x 10 5 cells/well , or Vero E6 1.25 x 10 5 cells/well cells seeded in 24-well plates and cultured overnight were infected MOI = 0.01 with the indicated rLCMVs. At the indicated times pi, TCSs were collected and virus titers determined by IFFA. Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP.",
"Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP. A549 cells were infected MOI = 0.1 with the indicated rLCMV or mock-infected, and 36 h later infected with SeV MOI = 1 . At 24 h pi with SeV, TCS were collected and used, following virus inactivation by U.V., to treat Vero E6 cells for 16 h, followed by infection with rVSV MOI = 0.1 rVSV + or mockinfection rVSV - . At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production.",
"At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production. ATP1A1 or with NC siRNA 72 h prior to infection with rLCMV/ZsG. We found that siRNAmediated kd of ATP1A1 dramatically inhibited ZsGreen expression Fig 3Ci , which was associated with a significant reduction of infectious LCMV progeny Fig 3Cii . Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both.",
"Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both. Another consideration is that only a minor fraction of NP might be engaged in the interaction with a given host cell protein, and therefore, detection of these interactions would require highly sensitive methods such as LC-MS/MS. To overcome this problem we used confocal microscopy to examine the co-localization of NP with ATP1A1 and PHB in LCMV-infected cells. Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries .",
"Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries . The PHB inhibitor rocaglamide is a flavagline from an Aglaia tree used in traditional Chinese medicine that has potent anticancer activity . To examine whether pharmacological inhibition of ATP1A1 or PHB inhibited LCMV multiplication, we pretreated human A549 and HEK 293T , nonhuman primate Vero E6 , and rodent murine L929 and hamster BHK-21 cells with ouabain or rocaglamide and infected them with rLCMV/eGFP S1 Fig . Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition .",
"Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition . Likewise, we observed a dose-dependent rocaglamide inhibition of eGFP expression in all cell lines infected with rLCMV/eGFP S1B Fig . Consistent with these findings, production of infectious LCMV progeny was reduced by treatment with either ouabain or rocaglamide Fig 5A within a concentration range that had minimal impact on cell viability Fig 5B . To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability.",
"To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability. To gain insights about the mechanisms by which ouabain and rocaglamide exert their anti-LCMV activity, we examined effects of these compounds on distinct steps of the LCMV life cycle. First, we asked whether ouabain and rocaglamide affected cell entry of LCMV. We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection.",
"We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection. The timing of compound addition did not significantly change the number of eGFP-positive cells, indicating that neither ouabain nor rocaglamide inhibited cell entry of LCMV. The number of eGFP + cells in ouabain-treated cells was reduced at all time-of-addition points compared to vehicle dimethyl sulfoxide DMSO -treated cells, but was similar to that observed in ammonium chloride-treated cells. Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription.",
"Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription. To further investigate the effect of ouabain and rocaglamide on virus RNA synthesis, we infected A549 cells with a recombinant single-cycle infectious LCMV expressing eGFP rLCMVΔGPC/eGFP and treated cells with either ouabain or rocaglamide. Seventy-two hours later, we determined percent normalized eGFP expression in infected cells Fig 6B . Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis.",
"Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis. We also examined the effect of ouabain and rocaglamide on the late step of the arenavirus life cycle, Z-mediated budding. For this experiment, we transfected cells with Z-Strep-and Z-FLAG DYKDDDDK epitope -expressing plasmids from LCMV and LASV, respectively. At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide.",
"At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide. At 24 h, we determined by WB levels of Z protein in both whole cell lysates and associated with virus-like particles VLPs collected from TCS. Treatment with rocaglamide, but not with ouabain, caused a reduction in both LCMV and LASV Z budding efficiency Fig 6C and 6D . The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments.",
"The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments. The first experiment involved the use of a newly generated single-cycle infectious recombinant LCMV expressing the reporter gene ZsGreen scrLCMV/ZsG-P2A-NP to infect MOI = 0.1 A549 cells 1 st infection . These cells were subsequently transfected with a plasmid expressing LCMV GPC. After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F .",
"After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F . The second experiment involved infection MOI = 0.1 of cells with WT LCMV, and 48 h later we washed infected cells three times to remove the extracellular infectious progeny produced during the first 48 h of infection. Then, fresh media containing ouabain or DMSO vehicle control were added, and 24 h later we determined virus titers in TCS Fig 6G . Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections.",
"Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections. Since ouabain and rocaglamide inhibited different steps of the LCMV life cycle, we examined whether their use in combination results in a synergistic anti-LCMV effect. For this experiment, we infected A549 cells with rLCMV/eGFP and treated them with ouabain and rocaglamide using different concentrations and combinations. At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide.",
"At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide. We next asked whether the ATP1A1 and PHB host-cell factors contributed also to multiplication of viral hemorrhagic fever-causing LASV. We treated A549 cells with ouabain, bufalin, or rocaglamide and inoculated the treated cells with recombinant LASV expressing eGFP rLASV/eGFP . eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 .",
"eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 . We also tested the effect of compounds targeting ATP1A1 and PHB on multiplication of JUNV. Consistent with our results obtained with LCMV and LASV, ouabain, bufalin, and rocaglamide strongly suppressed JUNV multiplication S3 Fig . These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses.",
"These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses. Our approach using a recombinant LCMV expressing NP with an affinity tag facilitated defining the NP interactome in the context of LCMV infection. Recently, using an NP-specific monoclonal antibody mAb to precipitate NP and associated cellular protein partners in a mammarenavirus NP interactome, King et al. identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al.",
"identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al. Differences in both experimental conditions and analysis methodologies used to generate the LCMV NP interactome documented by King et al. and ours likely h post-transfection, cells were washed with fresh media to eliminate Z-mediated production of VLPs in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain or Roc-A at the indicated concentrations. VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D .",
"VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D . Budding efficiency for each sample was estimated by dividing the signal intensity of the Z protein associated with VLPs by that of Z detected in the cell lysate. Numbers on the bottom of panel C correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments.",
"Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments. F Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected MOI = 0.1 with scrLCMV/ZsG 1 st infection for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared.",
"At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. ZsGreen signal intensity was measured by a fluorescent plate reader. GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%.",
"GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%. Data represent means ± SD from three independent experiments. contributed to the observed differences between data sets. Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems.",
"Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems. We used results of the eGFP-Strep interactome, determined in r3LCMV/eGFP-Strep-infected cells, as a control to filter out non-specific NP interactions, which may have resulted in a higher degree of stringency than in the study by King et al for selection of NP-interacting candidates. The combined information provided by the NP interactome reported by King et al. and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction .",
"and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction . The anti-IFN activity of NP was mapped to its C-terminal part and linked to the 3'-5' exonuclease domain present with the NP C-terminal part . Inhibitor-B kinase ε IKKε was identified as an NP-binding protein using plasmid-mediated overexpression in transfected cells , and the NP-IKKε binding affinity correlated with NP's ability to inhibit IFN-I induction . We, as well as the work by King et al. , did not detect this NP-IKKε interaction.",
"We, as well as the work by King et al. , did not detect this NP-IKKε interaction. This discrepancy may have been caused by very low expression of IKKε in LCMV-infected cells, which prevented detection of IKKε by LC-MS/MS. Alternatively, NP-IKKε interaction could possibility be temporarily regulated and take place at early times pi, but could be mostly absent at 48 h pi, the time at which we prepared the cell lysates for our proteomics studies. Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit .",
"Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit . ATP1A1 represents one of four α subunits . Recent evidence has suggested that the Na + /K + -ATPase is involved in multiple cell signaling pathways that are independent of its ion-pumping function . Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase .",
"Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase . Thus, a low concentration of ouabain induces a conformational change in ATP1A1 that results in activation and release of proto-oncogene tyrosine protein kinase, Src, from ATP1A1, followed by activation of as yet unknown downstream signaling that inhibits, for instance, cell entry of murine hepatitis virus MHV . However, our results indicated that ouabain did not interfere with LCMV cell entry. In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV.",
"In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV. Moreover, cell entry of JUNV occurs also by clathrin-mediated endocytosis , a process affected by Src signaling. Ouabain has been clinically used in several European countries for the management of congestive heart failure, whereas bufalin has been tested in clinical trials for cancer treatments , and the CST digoxin has been FDA-approved since 1997 to treat heart failure and atrial fibrillation. Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry.",
"Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry. In contrast, PHB was reported to be a cell entry receptor for DENV-2 and CHIKV . On the other hand, PHB did not act as a virus cell entry receptor for HCV. Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV .",
"Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV . These findings indicate that PHB is involved in different steps of the life cycle of a variety of viruses, and thereby an attractive target for the development of broad-spectrum antiviral drugs. Rocaglate is a group of natural compounds, which includes rocaglamide, that inhibits protein synthesis by targeting the ATP-dependent DEAD-box RNA helicase eukaryotic initiation factor 4A eIF4A and exerts anti-tumor activity . The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication.",
"The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication. These proteins, which included MAP1B, might have anti-LCMV activity. MAP1B has been shown to bind to nonstructural proteins 1 NS1 and 2 NS2 of human respiratory syncytial virus HRSV . NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined .",
"NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined . Examining the role of NP-MAP1B interaction in modulating NP's ability to inhibit induction of type I IFN is of interest. We identified among the NP-interacting host cell proteins the RNA helicase Moloney leukemia virus 10 MOV10 , which has been reported to be an antiviral factor for FLUAV , retroviruses , and DENV-2 . We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions.",
"We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions. MOV10 was shown to enhance IRF-3-mediated IFN-I induction following SeV infection through a tank binding kinase 1 TBK1 -independent and IKKε-dependent manner. This finding was further supported by demonstrating MOV10-IKKε interaction by co-immunoprecipitation studies . We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined.",
"We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined. Several members of the mammalian chaperonin-containing T-complex CCT were identified as prominent hits in our NP interactome. The mammalian CCT is critical for folding of many proteins with important functions in diverse cellular processes , and may protect complex protein topologies within its central cavity during biosynthesis and folding . The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV .",
"The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV . However, the role of these CCT proteins in virus multiplication remains unknown and may involve functions other than acting as molecular chaperones. Previous studies documented the presence of several components of the of eIF4F, including 4A, within viral replication-transcription complexes RTC detected in cells infected with LCMV and TCRV . These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity.",
"These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity. In this work, we documented the generation of rLCMV/Strep-NP and its use to define the NP-interactome in infected cells. We presented evidence that ATP1A1 and PHB contribute to efficient multiplication of mammarenaviruses using genetics and pharmacological inhibition of the genes. Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins.",
"Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins. Future studies elucidating the roles of pro-and antiviral host-cell factors identified in this study in mammarenavirus multiplication will advance our understanding of the multiple functions of NP and uncover novel cellular targets for the development of antimammarenaviral drugs. In addition, by identifying proviral host-cell factors, drugs that are already approved can be repurposing as therapeutics to combat human pathogenic mammarenavirus infections. Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described .",
"Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described . Generation of rLCMV/NP D382A and SeV, strain Cantell, was described 30, 82, 83 . An rLCMV lacking GPC and expressing eGFP rLCMVΔGPC/eGFP was generated by reverse genetics using procedures previously described . rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B .",
"rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B . The rLCMV expressing eGFP rLCMV/eGFP was generated as described , and the rLCMV expressing ZsGreen rLCMV/ZsG instead of eGFP was generated by reverse genetics using similar procedures to generate rLCMV/eGFP. Generation of rLASV expressing eGFP rLASV/eGFP will be described elsewhere. A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG.",
"A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG. scrLCMV/ZsG-P2A-NP was rescued by reverse genetics using similar procedures to generate rLCMVΔGPC/eGFP . LCMV titers were determined by immunofocus forming assay IFFA as described . Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA .",
"Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA . VSV titers were determined by a plaque assay. Total cell lysates were prepared in PD lysis buffer + 250 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% TritonX-100, 10% glycerol, 1 mM of MgCl 2 , 1 μM of CaCl 2 , 1 μM of ZnCl 2 and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA .",
"Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA . To detect Strep-tagged proteins, membranes were reacted with mouse monoclonal antibodies to Strep QIAGEN, Germantown, MD , eGFP Takara Bio USA, Mountain View, CA , GP 2 We33/ 36 , ATP1A1 TehrmoFisher Scientific, Rockford, IL , PHB Abcam, Cambridge, MA or rabbit polyclonal antibodies to α-tubulin Cell Signaling Technologies, Danvers, MA or glyceraldehyde-3-phosphate dehydrogenase GAPDH, Millipore , respectively, followed by incubation with appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G IgG antibodies Jackson ImmunoResearch Laboratories, West Grove, PA . SuperSignal West Pico or Femto chemiluminescent substrate Thermo Fisher Scientific was used to elicit chemiluminescent signals that were visualized using ImageQuant LAS 4000 Imager GE Healthcare Bio-Sciences, Pittsburgh, PA . Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1.",
"Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1. At 48 h pi, cells were washed three times with ice-cold PBS, scraped into fresh ice-cold PBS, and centrifuged at 400 x g at 4˚C for 10 min. Supernatant was removed, and cells were lysed with 12 ml of PD lysis buffer + supplemented with halt protease and phosphatase inhibitor cocktail Thermo Fisher Scientific and 5 μg/ml of deoxyribonuclease I Worthington Biochemical Corporation, Lakewood, NJ . Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C.",
"Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C. After 2 h of incubation, the resin was washed three times with PD lysis buffer + and once with PD lysis buffer without TritonX-100 PD lysis buffer - . After the centrifugation at 1,600 x g and 4˚C for 5 min, the last wash buffer was removed, and protein complexes associated with the resin were eluted into 2 ml of PD lysis buffer - containing 2.5 mM of desthiobiotin. The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn.",
"The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn. A MudPIT microcolumn was prepared by first creating a Kasil frit at one end of an un-deactivated 250-μm outside diameter OD capillary tube interior diameter of 360 μm Agilent Technologies, Inc., Santa Clara, CA . The Kasil frit was prepared by briefly dipping a 20-30-cm capillary tube in 300 μl of Kasil 1624 potassium silicate well-mixed solution PQ Corporation, Malvern, PA and 100 μl of formamide, curing at 100˚C for 4 h, and cutting the frit to a length of %2 mm. Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading.",
"Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading. MudPIT analysis. An analytical reversed-phase liquid chromatography column was generated by pulling a 100-μm interior diameter ID of 360 μm OD capillary tube Polymicro Technologies, Phoenix, AZ to 5-μm ID tip. Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid .",
"Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid . MudPIT and analytical columns were assembled using a zero-dead volume union Upchurch Scientific, Oak Harbor, WA . LC-MS/MS analysis was performed with an Agilent high-pressure LC pump Agilent and linear quadrupole ion dual cell trap Orbitrap Velos Thermo using an in-house built electrospray stage. Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient.",
"Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient. Data analysis. Protein and peptide identification were performed with Integrated Proteomics Pipeline-IP2 Integrated Proteomics Applications, San Diego, CA. integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in.",
"integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in. Spectrum raw files were extracted into ms2 files from raw files using open source RawExtract 1.9.9 Scripps Research Institute, La Jolla, CA; and the tandem mass spectra were searched against a human protein database UniprotKB . To accurately estimate peptide probabilities and false discovery rates, we used a decoy database containing the reversed sequences of all the proteins appended to the target database. Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system.",
"Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system. The search space included half and fully tryptic peptide candidates that fell within the mass tolerance window with no miscleavage constraint. Carbamidomethylation +57.02146 Da of cysteine was considered as a static modification. siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG.",
"siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG. siRNA target host-cell proteins were selected based on availability of validated siRNA sequences. The siRNAs we used to examine the effects on LCMV multiplication of knockdown expression of NP-interacting host cell protein candidate hits corresponded to the Genome-wide ON TAR-GET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene; Dharmacon, Lafayette, CO . A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min.",
"A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. The total protein concentration of clarified cell lysate was measured by Pierce BCA Protein Assay Kit Thermo Fisher Scientific . The same amount of protein from each sample was subjected to SDS-PAGE, and the protein expression of siRNA-targeted genes was analyzed by western blots. Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI .",
"Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI . Green fluorescence eGFP or ZsGreen and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, Bio-Tek, Winooski, VT . Mock-and virus-infected cells were fixed with 4% PFA. After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488.",
"After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488. In some samples, primary antibody against ATP1A1 or PHB was omitted to determine background fluorescence. To visualize nuclei, DAPI Fluoromount-G SouthernBiotech, Birmingham, AL was used to mount coverslips on a slide glass. Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss .",
"Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss . Eight green NP-positive cells were marked and every pixel in the marked area was plotted in the scatter diagram based on its intensity level from each channel. Thresholds for green and red channels were determined using mock-infected cells stained with VL-4-AF488 anti-NP and anti-mouse IgG antibody conjugated with Alexa Fluor 568, without using anti-ATP1A1 or -PHB antibodies. Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels.",
"Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels. This calculation was repeated for eight individual cells. To assess the specificity of co-localization, we determined weighted CC by taking into consideration the brightness of each channel signal. Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software.",
"Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software. A549 or Vero E6 cells seeded 2.0 x 10 4 cells/well in a 96-well plate and cultured overnight were treated with 3-fold serial compound dilutions at 37˚C and 5% CO 2 for 2 h, followed by infection with rLCMV/eGFP MOI = 0.01 . Compounds were present to study endpoint. At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%.",
"At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%. The IC 50 concentrations were determined using GraphPad Prism. A549 or Vero E6 cells seeded in a 96-well plate 2.0 x 10 4 cells/well and cultured overnight were treated with 3-fold serial compound dilutions and cultured at 37˚C and 5% CO 2 for 48 h. Then, CellTiter 96 AQ ueous one solution reagent Promega, Madison, WI was added. Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%.",
"Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%. The CC 50 concentrations were determined using GraphPad Prism. A plasmid expressing C-terminus Strep-tagged Z protein pC-LCMV-Z-Strep was generated using similar procedure to generate a plasmid expressing C-terminus FLAG-tagged LASV Z protein pC-LASV-Z-FLAG , and the budding assay was performed as previously described . Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium.",
"Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium. After the removal of the last wash medium, cells were cultured in fresh medium containing ouabain 30 or 40 nM or rocaglamide 50 or 100 nM or equivalent concentration of DMSO, and 24 h later, virion-like particle VLP -containing TCS and cells were collected. Total cell lysate was prepared by lysing the cells with lysis buffer 1% NP-40, 50 mM of Tris-HCl pH 8.0 , 62.5 mM NaCl, 0.4% sodium deoxycholate . After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots.",
"After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots. A549 cells infected with rLCMV/eGFP were harvested using Accutase cell detachment solution Innovative Cell Technologies, San Diego, CA and fixed with 4% PFA in PBS. eGFP expression was examined by flow cytometry using a BD LSR II Becton Dickson , and data were analyzed with FlowJo Tree Star, Inc., Ashland, OR . 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO .",
"293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO . At 48 h pi, TCS was collected and used to infect fresh monolayers of BHK-21 cells seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . A549 cells seeded 2 x 10 5 cells/well in a 96-well plate and cultured overnight were treated with combinations of different concentrations of ouabain and rocaglamide for 2 h and then infected MOI = 0.01 with rLCMV/eGFP. Compounds were present in the culture medium throughout the experiment. At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . eGFP readouts were normalized by DAPI readouts, and normalized data were used to analyze synergistic effect of the two compounds by the MacSynergy II program . Data were analyzed for p values by a two-tailed unpaired t test using GraphPad Prism software."
] | 1,687 | 5,154 |
What drug is used to treat congestive heart failure? | ouabain | [
"Several mammalian arenaviruses mammarenaviruses cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus LCMV , is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors.",
"Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.",
"We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. Text: Introduction Mammarenaviruses Arenaviridae: Mammarenavirus cause chronic infections of rodents worldwide . Invasion of human dwellings by infected rodents can result in human infections through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW .",
"Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW . The OW Lassa virus LASV , causative agent of Lassa fever LF , is the most significant OW mammarenaviral pathogen. LASV is estimated to infect several hundred thousand individuals annually in Western Africa, resulting in a high number of LF cases associated with high morbidity and lethality. Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever .",
"Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever . The worldwide-distributed OW mammarenavirus lymphocytic choriomeningitis virus LCMV is a neglected human pathogen of clinical significance especially in congenital infections . Moreover, LCMV poses a particular threat to immunocompromised individuals, as has been illustrated by fatal cases of LCMV infection associated with organ transplants . However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina.",
"However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina. Likewise, current anti-mammarenavirus therapy is limited to an offlabel use of the nucleoside analogue ribavirin that is only partially effective and can cause significant side effects . Development of effective anti-mammarenavirus drugs has been hampered by the lack of detailed understanding of virus/host-cell interactions required for mammarenavirus multiplication that could represent amenable targets for antiviral therapy. the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B .",
"the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B . To facilitate the identification of specific PPI between NP and host cell proteins, we used our mammarenavirus tri-segmented r3 platform to design an r3LCMV expressing a C-terminus Strep-tag version of enhanced green fluorescent protein r3LCMV/eGFP-Strep that we used as a negative control Fig 1A and 1B . We rescued rLCMV/Strep-NP and r3LCMV/eGFP-Strep and confirmed the expression of strep-tagged NP and eGFP in rLCMV/ Strep-NP-and r3LCMV/eGFP-Strep-infected cells, respectively Fig 1C . Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV.",
"Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV. However, both rLCMV/Strep-NP and r3LCMV/eGFP-Strep had WT-like growth kinetics and reached high titers. As with WT LCMV, infection with rLCMV/Strep-NP prevented production of bioactive IFN-I by cells in response to Sendai virus SeV infection as determined using an IFN bioassay based on protection against the cytopathic effect CPE induced by infection with vesicular stomatitis virus VSV Fig 1E . Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE.",
"Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE. We selected the human A549 cell line because lung epithelial cells are one of the initial cell targets of humans following inhalation of mammarenavirions. We infected A549 cells multiplicity of infection MOI = 0.1 with either rLCMV/Strep-NP or r3LCMV/eGFP-Strep Fig 2A . At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining.",
"At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining. We compared the pattern of stained protein bands detected between rLCMV/Strep-NP-and r3LCMV/eGFP-Strepinfected samples and confirmed the presence of Strep-NP and eGFP-Strep in pertinent samples Fig 2B . Protein complexes in the rest of eluates from the PD samples were concentrated by trichloroacetic acid TCA precipitation and subjected to trypsin digestion Fig 2A . Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 .",
"Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 . Filtering the data using these criteria resulted in identification of 139 candidate host-cell proteins as NP-interacting partners. Among 53 proteins found present in both NP-and eGFP-PD samples, 36 had spectral counts in the NP-PD sample that were less than two-fold higher than their corresponding spectral counts in the eGFP-PD sample Fig 2C and S1 Table . Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV.",
"Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV. The protein annotation through evolutionary relationship PANTHER protein family classification Biological Process of the NP-interacting protein candidates revealed that a large number of proteins were involved in metabolic and cellular processes Fig 2D . We also analyzed the molecular functions of the NP-interacting protein candidates according to the PAN-THER protein profile classification Fig 2E , which revealed diversified biochemical functions enriched for nucleic acid-binding proteins and chaperones. To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene .",
"To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene . OTPs are latest generation of siRNAs and offer significant advantages over previous generations. Off-target effects are primarily driven by antisense strand microRNA miR -like seed activity. In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy.",
"In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy. Numerous host-cell factors showed an anti-LCMV effect increased ZsG expression by kd of the genes , including microtubule-associated protein 1B MAP1B , dengue virus 2 DENV-2 , and chikungunya virus CHIKV . To begin assessing the potential biological implications of the identified NP-host cell protein interactions, we selected ATP1A1 and PHB given the availability of reagents, existing knowledge about their roles in cell physiology, and evidence of participation in multiplication of other viruses. We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers.",
"We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers. For this, we transfected A549 cells with siRNA targeting Expression of Strep-tagged proteins. A549 cells seeded 5.0 x 10 5 cells/well in a 6-well plate and cultured overnight were infected MOI = 0.1 with the indicated rLCMVs. At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins.",
"At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins. BHK-21 1.75 x 10 5 cells/well , A549 1.25 x 10 5 cells/well , or Vero E6 1.25 x 10 5 cells/well cells seeded in 24-well plates and cultured overnight were infected MOI = 0.01 with the indicated rLCMVs. At the indicated times pi, TCSs were collected and virus titers determined by IFFA. Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP.",
"Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP. A549 cells were infected MOI = 0.1 with the indicated rLCMV or mock-infected, and 36 h later infected with SeV MOI = 1 . At 24 h pi with SeV, TCS were collected and used, following virus inactivation by U.V., to treat Vero E6 cells for 16 h, followed by infection with rVSV MOI = 0.1 rVSV + or mockinfection rVSV - . At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production.",
"At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production. ATP1A1 or with NC siRNA 72 h prior to infection with rLCMV/ZsG. We found that siRNAmediated kd of ATP1A1 dramatically inhibited ZsGreen expression Fig 3Ci , which was associated with a significant reduction of infectious LCMV progeny Fig 3Cii . Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both.",
"Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both. Another consideration is that only a minor fraction of NP might be engaged in the interaction with a given host cell protein, and therefore, detection of these interactions would require highly sensitive methods such as LC-MS/MS. To overcome this problem we used confocal microscopy to examine the co-localization of NP with ATP1A1 and PHB in LCMV-infected cells. Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries .",
"Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries . The PHB inhibitor rocaglamide is a flavagline from an Aglaia tree used in traditional Chinese medicine that has potent anticancer activity . To examine whether pharmacological inhibition of ATP1A1 or PHB inhibited LCMV multiplication, we pretreated human A549 and HEK 293T , nonhuman primate Vero E6 , and rodent murine L929 and hamster BHK-21 cells with ouabain or rocaglamide and infected them with rLCMV/eGFP S1 Fig . Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition .",
"Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition . Likewise, we observed a dose-dependent rocaglamide inhibition of eGFP expression in all cell lines infected with rLCMV/eGFP S1B Fig . Consistent with these findings, production of infectious LCMV progeny was reduced by treatment with either ouabain or rocaglamide Fig 5A within a concentration range that had minimal impact on cell viability Fig 5B . To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability.",
"To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability. To gain insights about the mechanisms by which ouabain and rocaglamide exert their anti-LCMV activity, we examined effects of these compounds on distinct steps of the LCMV life cycle. First, we asked whether ouabain and rocaglamide affected cell entry of LCMV. We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection.",
"We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection. The timing of compound addition did not significantly change the number of eGFP-positive cells, indicating that neither ouabain nor rocaglamide inhibited cell entry of LCMV. The number of eGFP + cells in ouabain-treated cells was reduced at all time-of-addition points compared to vehicle dimethyl sulfoxide DMSO -treated cells, but was similar to that observed in ammonium chloride-treated cells. Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription.",
"Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription. To further investigate the effect of ouabain and rocaglamide on virus RNA synthesis, we infected A549 cells with a recombinant single-cycle infectious LCMV expressing eGFP rLCMVΔGPC/eGFP and treated cells with either ouabain or rocaglamide. Seventy-two hours later, we determined percent normalized eGFP expression in infected cells Fig 6B . Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis.",
"Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis. We also examined the effect of ouabain and rocaglamide on the late step of the arenavirus life cycle, Z-mediated budding. For this experiment, we transfected cells with Z-Strep-and Z-FLAG DYKDDDDK epitope -expressing plasmids from LCMV and LASV, respectively. At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide.",
"At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide. At 24 h, we determined by WB levels of Z protein in both whole cell lysates and associated with virus-like particles VLPs collected from TCS. Treatment with rocaglamide, but not with ouabain, caused a reduction in both LCMV and LASV Z budding efficiency Fig 6C and 6D . The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments.",
"The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments. The first experiment involved the use of a newly generated single-cycle infectious recombinant LCMV expressing the reporter gene ZsGreen scrLCMV/ZsG-P2A-NP to infect MOI = 0.1 A549 cells 1 st infection . These cells were subsequently transfected with a plasmid expressing LCMV GPC. After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F .",
"After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F . The second experiment involved infection MOI = 0.1 of cells with WT LCMV, and 48 h later we washed infected cells three times to remove the extracellular infectious progeny produced during the first 48 h of infection. Then, fresh media containing ouabain or DMSO vehicle control were added, and 24 h later we determined virus titers in TCS Fig 6G . Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections.",
"Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections. Since ouabain and rocaglamide inhibited different steps of the LCMV life cycle, we examined whether their use in combination results in a synergistic anti-LCMV effect. For this experiment, we infected A549 cells with rLCMV/eGFP and treated them with ouabain and rocaglamide using different concentrations and combinations. At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide.",
"At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide. We next asked whether the ATP1A1 and PHB host-cell factors contributed also to multiplication of viral hemorrhagic fever-causing LASV. We treated A549 cells with ouabain, bufalin, or rocaglamide and inoculated the treated cells with recombinant LASV expressing eGFP rLASV/eGFP . eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 .",
"eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 . We also tested the effect of compounds targeting ATP1A1 and PHB on multiplication of JUNV. Consistent with our results obtained with LCMV and LASV, ouabain, bufalin, and rocaglamide strongly suppressed JUNV multiplication S3 Fig . These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses.",
"These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses. Our approach using a recombinant LCMV expressing NP with an affinity tag facilitated defining the NP interactome in the context of LCMV infection. Recently, using an NP-specific monoclonal antibody mAb to precipitate NP and associated cellular protein partners in a mammarenavirus NP interactome, King et al. identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al.",
"identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al. Differences in both experimental conditions and analysis methodologies used to generate the LCMV NP interactome documented by King et al. and ours likely h post-transfection, cells were washed with fresh media to eliminate Z-mediated production of VLPs in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain or Roc-A at the indicated concentrations. VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D .",
"VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D . Budding efficiency for each sample was estimated by dividing the signal intensity of the Z protein associated with VLPs by that of Z detected in the cell lysate. Numbers on the bottom of panel C correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments.",
"Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments. F Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected MOI = 0.1 with scrLCMV/ZsG 1 st infection for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared.",
"At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. ZsGreen signal intensity was measured by a fluorescent plate reader. GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%.",
"GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%. Data represent means ± SD from three independent experiments. contributed to the observed differences between data sets. Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems.",
"Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems. We used results of the eGFP-Strep interactome, determined in r3LCMV/eGFP-Strep-infected cells, as a control to filter out non-specific NP interactions, which may have resulted in a higher degree of stringency than in the study by King et al for selection of NP-interacting candidates. The combined information provided by the NP interactome reported by King et al. and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction .",
"and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction . The anti-IFN activity of NP was mapped to its C-terminal part and linked to the 3'-5' exonuclease domain present with the NP C-terminal part . Inhibitor-B kinase ε IKKε was identified as an NP-binding protein using plasmid-mediated overexpression in transfected cells , and the NP-IKKε binding affinity correlated with NP's ability to inhibit IFN-I induction . We, as well as the work by King et al. , did not detect this NP-IKKε interaction.",
"We, as well as the work by King et al. , did not detect this NP-IKKε interaction. This discrepancy may have been caused by very low expression of IKKε in LCMV-infected cells, which prevented detection of IKKε by LC-MS/MS. Alternatively, NP-IKKε interaction could possibility be temporarily regulated and take place at early times pi, but could be mostly absent at 48 h pi, the time at which we prepared the cell lysates for our proteomics studies. Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit .",
"Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit . ATP1A1 represents one of four α subunits . Recent evidence has suggested that the Na + /K + -ATPase is involved in multiple cell signaling pathways that are independent of its ion-pumping function . Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase .",
"Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase . Thus, a low concentration of ouabain induces a conformational change in ATP1A1 that results in activation and release of proto-oncogene tyrosine protein kinase, Src, from ATP1A1, followed by activation of as yet unknown downstream signaling that inhibits, for instance, cell entry of murine hepatitis virus MHV . However, our results indicated that ouabain did not interfere with LCMV cell entry. In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV.",
"In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV. Moreover, cell entry of JUNV occurs also by clathrin-mediated endocytosis , a process affected by Src signaling. Ouabain has been clinically used in several European countries for the management of congestive heart failure, whereas bufalin has been tested in clinical trials for cancer treatments , and the CST digoxin has been FDA-approved since 1997 to treat heart failure and atrial fibrillation. Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry.",
"Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry. In contrast, PHB was reported to be a cell entry receptor for DENV-2 and CHIKV . On the other hand, PHB did not act as a virus cell entry receptor for HCV. Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV .",
"Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV . These findings indicate that PHB is involved in different steps of the life cycle of a variety of viruses, and thereby an attractive target for the development of broad-spectrum antiviral drugs. Rocaglate is a group of natural compounds, which includes rocaglamide, that inhibits protein synthesis by targeting the ATP-dependent DEAD-box RNA helicase eukaryotic initiation factor 4A eIF4A and exerts anti-tumor activity . The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication.",
"The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication. These proteins, which included MAP1B, might have anti-LCMV activity. MAP1B has been shown to bind to nonstructural proteins 1 NS1 and 2 NS2 of human respiratory syncytial virus HRSV . NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined .",
"NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined . Examining the role of NP-MAP1B interaction in modulating NP's ability to inhibit induction of type I IFN is of interest. We identified among the NP-interacting host cell proteins the RNA helicase Moloney leukemia virus 10 MOV10 , which has been reported to be an antiviral factor for FLUAV , retroviruses , and DENV-2 . We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions.",
"We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions. MOV10 was shown to enhance IRF-3-mediated IFN-I induction following SeV infection through a tank binding kinase 1 TBK1 -independent and IKKε-dependent manner. This finding was further supported by demonstrating MOV10-IKKε interaction by co-immunoprecipitation studies . We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined.",
"We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined. Several members of the mammalian chaperonin-containing T-complex CCT were identified as prominent hits in our NP interactome. The mammalian CCT is critical for folding of many proteins with important functions in diverse cellular processes , and may protect complex protein topologies within its central cavity during biosynthesis and folding . The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV .",
"The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV . However, the role of these CCT proteins in virus multiplication remains unknown and may involve functions other than acting as molecular chaperones. Previous studies documented the presence of several components of the of eIF4F, including 4A, within viral replication-transcription complexes RTC detected in cells infected with LCMV and TCRV . These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity.",
"These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity. In this work, we documented the generation of rLCMV/Strep-NP and its use to define the NP-interactome in infected cells. We presented evidence that ATP1A1 and PHB contribute to efficient multiplication of mammarenaviruses using genetics and pharmacological inhibition of the genes. Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins.",
"Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins. Future studies elucidating the roles of pro-and antiviral host-cell factors identified in this study in mammarenavirus multiplication will advance our understanding of the multiple functions of NP and uncover novel cellular targets for the development of antimammarenaviral drugs. In addition, by identifying proviral host-cell factors, drugs that are already approved can be repurposing as therapeutics to combat human pathogenic mammarenavirus infections. Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described .",
"Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described . Generation of rLCMV/NP D382A and SeV, strain Cantell, was described 30, 82, 83 . An rLCMV lacking GPC and expressing eGFP rLCMVΔGPC/eGFP was generated by reverse genetics using procedures previously described . rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B .",
"rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B . The rLCMV expressing eGFP rLCMV/eGFP was generated as described , and the rLCMV expressing ZsGreen rLCMV/ZsG instead of eGFP was generated by reverse genetics using similar procedures to generate rLCMV/eGFP. Generation of rLASV expressing eGFP rLASV/eGFP will be described elsewhere. A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG.",
"A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG. scrLCMV/ZsG-P2A-NP was rescued by reverse genetics using similar procedures to generate rLCMVΔGPC/eGFP . LCMV titers were determined by immunofocus forming assay IFFA as described . Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA .",
"Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA . VSV titers were determined by a plaque assay. Total cell lysates were prepared in PD lysis buffer + 250 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% TritonX-100, 10% glycerol, 1 mM of MgCl 2 , 1 μM of CaCl 2 , 1 μM of ZnCl 2 and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA .",
"Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA . To detect Strep-tagged proteins, membranes were reacted with mouse monoclonal antibodies to Strep QIAGEN, Germantown, MD , eGFP Takara Bio USA, Mountain View, CA , GP 2 We33/ 36 , ATP1A1 TehrmoFisher Scientific, Rockford, IL , PHB Abcam, Cambridge, MA or rabbit polyclonal antibodies to α-tubulin Cell Signaling Technologies, Danvers, MA or glyceraldehyde-3-phosphate dehydrogenase GAPDH, Millipore , respectively, followed by incubation with appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G IgG antibodies Jackson ImmunoResearch Laboratories, West Grove, PA . SuperSignal West Pico or Femto chemiluminescent substrate Thermo Fisher Scientific was used to elicit chemiluminescent signals that were visualized using ImageQuant LAS 4000 Imager GE Healthcare Bio-Sciences, Pittsburgh, PA . Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1.",
"Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1. At 48 h pi, cells were washed three times with ice-cold PBS, scraped into fresh ice-cold PBS, and centrifuged at 400 x g at 4˚C for 10 min. Supernatant was removed, and cells were lysed with 12 ml of PD lysis buffer + supplemented with halt protease and phosphatase inhibitor cocktail Thermo Fisher Scientific and 5 μg/ml of deoxyribonuclease I Worthington Biochemical Corporation, Lakewood, NJ . Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C.",
"Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C. After 2 h of incubation, the resin was washed three times with PD lysis buffer + and once with PD lysis buffer without TritonX-100 PD lysis buffer - . After the centrifugation at 1,600 x g and 4˚C for 5 min, the last wash buffer was removed, and protein complexes associated with the resin were eluted into 2 ml of PD lysis buffer - containing 2.5 mM of desthiobiotin. The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn.",
"The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn. A MudPIT microcolumn was prepared by first creating a Kasil frit at one end of an un-deactivated 250-μm outside diameter OD capillary tube interior diameter of 360 μm Agilent Technologies, Inc., Santa Clara, CA . The Kasil frit was prepared by briefly dipping a 20-30-cm capillary tube in 300 μl of Kasil 1624 potassium silicate well-mixed solution PQ Corporation, Malvern, PA and 100 μl of formamide, curing at 100˚C for 4 h, and cutting the frit to a length of %2 mm. Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading.",
"Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading. MudPIT analysis. An analytical reversed-phase liquid chromatography column was generated by pulling a 100-μm interior diameter ID of 360 μm OD capillary tube Polymicro Technologies, Phoenix, AZ to 5-μm ID tip. Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid .",
"Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid . MudPIT and analytical columns were assembled using a zero-dead volume union Upchurch Scientific, Oak Harbor, WA . LC-MS/MS analysis was performed with an Agilent high-pressure LC pump Agilent and linear quadrupole ion dual cell trap Orbitrap Velos Thermo using an in-house built electrospray stage. Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient.",
"Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient. Data analysis. Protein and peptide identification were performed with Integrated Proteomics Pipeline-IP2 Integrated Proteomics Applications, San Diego, CA. integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in.",
"integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in. Spectrum raw files were extracted into ms2 files from raw files using open source RawExtract 1.9.9 Scripps Research Institute, La Jolla, CA; and the tandem mass spectra were searched against a human protein database UniprotKB . To accurately estimate peptide probabilities and false discovery rates, we used a decoy database containing the reversed sequences of all the proteins appended to the target database. Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system.",
"Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system. The search space included half and fully tryptic peptide candidates that fell within the mass tolerance window with no miscleavage constraint. Carbamidomethylation +57.02146 Da of cysteine was considered as a static modification. siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG.",
"siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG. siRNA target host-cell proteins were selected based on availability of validated siRNA sequences. The siRNAs we used to examine the effects on LCMV multiplication of knockdown expression of NP-interacting host cell protein candidate hits corresponded to the Genome-wide ON TAR-GET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene; Dharmacon, Lafayette, CO . A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min.",
"A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. The total protein concentration of clarified cell lysate was measured by Pierce BCA Protein Assay Kit Thermo Fisher Scientific . The same amount of protein from each sample was subjected to SDS-PAGE, and the protein expression of siRNA-targeted genes was analyzed by western blots. Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI .",
"Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI . Green fluorescence eGFP or ZsGreen and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, Bio-Tek, Winooski, VT . Mock-and virus-infected cells were fixed with 4% PFA. After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488.",
"After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488. In some samples, primary antibody against ATP1A1 or PHB was omitted to determine background fluorescence. To visualize nuclei, DAPI Fluoromount-G SouthernBiotech, Birmingham, AL was used to mount coverslips on a slide glass. Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss .",
"Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss . Eight green NP-positive cells were marked and every pixel in the marked area was plotted in the scatter diagram based on its intensity level from each channel. Thresholds for green and red channels were determined using mock-infected cells stained with VL-4-AF488 anti-NP and anti-mouse IgG antibody conjugated with Alexa Fluor 568, without using anti-ATP1A1 or -PHB antibodies. Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels.",
"Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels. This calculation was repeated for eight individual cells. To assess the specificity of co-localization, we determined weighted CC by taking into consideration the brightness of each channel signal. Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software.",
"Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software. A549 or Vero E6 cells seeded 2.0 x 10 4 cells/well in a 96-well plate and cultured overnight were treated with 3-fold serial compound dilutions at 37˚C and 5% CO 2 for 2 h, followed by infection with rLCMV/eGFP MOI = 0.01 . Compounds were present to study endpoint. At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%.",
"At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%. The IC 50 concentrations were determined using GraphPad Prism. A549 or Vero E6 cells seeded in a 96-well plate 2.0 x 10 4 cells/well and cultured overnight were treated with 3-fold serial compound dilutions and cultured at 37˚C and 5% CO 2 for 48 h. Then, CellTiter 96 AQ ueous one solution reagent Promega, Madison, WI was added. Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%.",
"Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%. The CC 50 concentrations were determined using GraphPad Prism. A plasmid expressing C-terminus Strep-tagged Z protein pC-LCMV-Z-Strep was generated using similar procedure to generate a plasmid expressing C-terminus FLAG-tagged LASV Z protein pC-LASV-Z-FLAG , and the budding assay was performed as previously described . Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium.",
"Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium. After the removal of the last wash medium, cells were cultured in fresh medium containing ouabain 30 or 40 nM or rocaglamide 50 or 100 nM or equivalent concentration of DMSO, and 24 h later, virion-like particle VLP -containing TCS and cells were collected. Total cell lysate was prepared by lysing the cells with lysis buffer 1% NP-40, 50 mM of Tris-HCl pH 8.0 , 62.5 mM NaCl, 0.4% sodium deoxycholate . After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots.",
"After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots. A549 cells infected with rLCMV/eGFP were harvested using Accutase cell detachment solution Innovative Cell Technologies, San Diego, CA and fixed with 4% PFA in PBS. eGFP expression was examined by flow cytometry using a BD LSR II Becton Dickson , and data were analyzed with FlowJo Tree Star, Inc., Ashland, OR . 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO .",
"293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO . At 48 h pi, TCS was collected and used to infect fresh monolayers of BHK-21 cells seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . A549 cells seeded 2 x 10 5 cells/well in a 96-well plate and cultured overnight were treated with combinations of different concentrations of ouabain and rocaglamide for 2 h and then infected MOI = 0.01 with rLCMV/eGFP. Compounds were present in the culture medium throughout the experiment. At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . eGFP readouts were normalized by DAPI readouts, and normalized data were used to analyze synergistic effect of the two compounds by the MacSynergy II program . Data were analyzed for p values by a two-tailed unpaired t test using GraphPad Prism software."
] | 1,687 | 5,155 |
What does ouabain inhibit? | ATP1A1 | [
"Several mammalian arenaviruses mammarenaviruses cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus LCMV , is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors.",
"Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.",
"We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. Text: Introduction Mammarenaviruses Arenaviridae: Mammarenavirus cause chronic infections of rodents worldwide . Invasion of human dwellings by infected rodents can result in human infections through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW .",
"Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW . The OW Lassa virus LASV , causative agent of Lassa fever LF , is the most significant OW mammarenaviral pathogen. LASV is estimated to infect several hundred thousand individuals annually in Western Africa, resulting in a high number of LF cases associated with high morbidity and lethality. Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever .",
"Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever . The worldwide-distributed OW mammarenavirus lymphocytic choriomeningitis virus LCMV is a neglected human pathogen of clinical significance especially in congenital infections . Moreover, LCMV poses a particular threat to immunocompromised individuals, as has been illustrated by fatal cases of LCMV infection associated with organ transplants . However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina.",
"However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina. Likewise, current anti-mammarenavirus therapy is limited to an offlabel use of the nucleoside analogue ribavirin that is only partially effective and can cause significant side effects . Development of effective anti-mammarenavirus drugs has been hampered by the lack of detailed understanding of virus/host-cell interactions required for mammarenavirus multiplication that could represent amenable targets for antiviral therapy. the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B .",
"the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B . To facilitate the identification of specific PPI between NP and host cell proteins, we used our mammarenavirus tri-segmented r3 platform to design an r3LCMV expressing a C-terminus Strep-tag version of enhanced green fluorescent protein r3LCMV/eGFP-Strep that we used as a negative control Fig 1A and 1B . We rescued rLCMV/Strep-NP and r3LCMV/eGFP-Strep and confirmed the expression of strep-tagged NP and eGFP in rLCMV/ Strep-NP-and r3LCMV/eGFP-Strep-infected cells, respectively Fig 1C . Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV.",
"Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV. However, both rLCMV/Strep-NP and r3LCMV/eGFP-Strep had WT-like growth kinetics and reached high titers. As with WT LCMV, infection with rLCMV/Strep-NP prevented production of bioactive IFN-I by cells in response to Sendai virus SeV infection as determined using an IFN bioassay based on protection against the cytopathic effect CPE induced by infection with vesicular stomatitis virus VSV Fig 1E . Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE.",
"Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE. We selected the human A549 cell line because lung epithelial cells are one of the initial cell targets of humans following inhalation of mammarenavirions. We infected A549 cells multiplicity of infection MOI = 0.1 with either rLCMV/Strep-NP or r3LCMV/eGFP-Strep Fig 2A . At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining.",
"At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining. We compared the pattern of stained protein bands detected between rLCMV/Strep-NP-and r3LCMV/eGFP-Strepinfected samples and confirmed the presence of Strep-NP and eGFP-Strep in pertinent samples Fig 2B . Protein complexes in the rest of eluates from the PD samples were concentrated by trichloroacetic acid TCA precipitation and subjected to trypsin digestion Fig 2A . Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 .",
"Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 . Filtering the data using these criteria resulted in identification of 139 candidate host-cell proteins as NP-interacting partners. Among 53 proteins found present in both NP-and eGFP-PD samples, 36 had spectral counts in the NP-PD sample that were less than two-fold higher than their corresponding spectral counts in the eGFP-PD sample Fig 2C and S1 Table . Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV.",
"Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV. The protein annotation through evolutionary relationship PANTHER protein family classification Biological Process of the NP-interacting protein candidates revealed that a large number of proteins were involved in metabolic and cellular processes Fig 2D . We also analyzed the molecular functions of the NP-interacting protein candidates according to the PAN-THER protein profile classification Fig 2E , which revealed diversified biochemical functions enriched for nucleic acid-binding proteins and chaperones. To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene .",
"To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene . OTPs are latest generation of siRNAs and offer significant advantages over previous generations. Off-target effects are primarily driven by antisense strand microRNA miR -like seed activity. In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy.",
"In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy. Numerous host-cell factors showed an anti-LCMV effect increased ZsG expression by kd of the genes , including microtubule-associated protein 1B MAP1B , dengue virus 2 DENV-2 , and chikungunya virus CHIKV . To begin assessing the potential biological implications of the identified NP-host cell protein interactions, we selected ATP1A1 and PHB given the availability of reagents, existing knowledge about their roles in cell physiology, and evidence of participation in multiplication of other viruses. We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers.",
"We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers. For this, we transfected A549 cells with siRNA targeting Expression of Strep-tagged proteins. A549 cells seeded 5.0 x 10 5 cells/well in a 6-well plate and cultured overnight were infected MOI = 0.1 with the indicated rLCMVs. At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins.",
"At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins. BHK-21 1.75 x 10 5 cells/well , A549 1.25 x 10 5 cells/well , or Vero E6 1.25 x 10 5 cells/well cells seeded in 24-well plates and cultured overnight were infected MOI = 0.01 with the indicated rLCMVs. At the indicated times pi, TCSs were collected and virus titers determined by IFFA. Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP.",
"Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP. A549 cells were infected MOI = 0.1 with the indicated rLCMV or mock-infected, and 36 h later infected with SeV MOI = 1 . At 24 h pi with SeV, TCS were collected and used, following virus inactivation by U.V., to treat Vero E6 cells for 16 h, followed by infection with rVSV MOI = 0.1 rVSV + or mockinfection rVSV - . At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production.",
"At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production. ATP1A1 or with NC siRNA 72 h prior to infection with rLCMV/ZsG. We found that siRNAmediated kd of ATP1A1 dramatically inhibited ZsGreen expression Fig 3Ci , which was associated with a significant reduction of infectious LCMV progeny Fig 3Cii . Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both.",
"Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both. Another consideration is that only a minor fraction of NP might be engaged in the interaction with a given host cell protein, and therefore, detection of these interactions would require highly sensitive methods such as LC-MS/MS. To overcome this problem we used confocal microscopy to examine the co-localization of NP with ATP1A1 and PHB in LCMV-infected cells. Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries .",
"Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries . The PHB inhibitor rocaglamide is a flavagline from an Aglaia tree used in traditional Chinese medicine that has potent anticancer activity . To examine whether pharmacological inhibition of ATP1A1 or PHB inhibited LCMV multiplication, we pretreated human A549 and HEK 293T , nonhuman primate Vero E6 , and rodent murine L929 and hamster BHK-21 cells with ouabain or rocaglamide and infected them with rLCMV/eGFP S1 Fig . Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition .",
"Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition . Likewise, we observed a dose-dependent rocaglamide inhibition of eGFP expression in all cell lines infected with rLCMV/eGFP S1B Fig . Consistent with these findings, production of infectious LCMV progeny was reduced by treatment with either ouabain or rocaglamide Fig 5A within a concentration range that had minimal impact on cell viability Fig 5B . To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability.",
"To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability. To gain insights about the mechanisms by which ouabain and rocaglamide exert their anti-LCMV activity, we examined effects of these compounds on distinct steps of the LCMV life cycle. First, we asked whether ouabain and rocaglamide affected cell entry of LCMV. We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection.",
"We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection. The timing of compound addition did not significantly change the number of eGFP-positive cells, indicating that neither ouabain nor rocaglamide inhibited cell entry of LCMV. The number of eGFP + cells in ouabain-treated cells was reduced at all time-of-addition points compared to vehicle dimethyl sulfoxide DMSO -treated cells, but was similar to that observed in ammonium chloride-treated cells. Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription.",
"Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription. To further investigate the effect of ouabain and rocaglamide on virus RNA synthesis, we infected A549 cells with a recombinant single-cycle infectious LCMV expressing eGFP rLCMVΔGPC/eGFP and treated cells with either ouabain or rocaglamide. Seventy-two hours later, we determined percent normalized eGFP expression in infected cells Fig 6B . Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis.",
"Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis. We also examined the effect of ouabain and rocaglamide on the late step of the arenavirus life cycle, Z-mediated budding. For this experiment, we transfected cells with Z-Strep-and Z-FLAG DYKDDDDK epitope -expressing plasmids from LCMV and LASV, respectively. At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide.",
"At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide. At 24 h, we determined by WB levels of Z protein in both whole cell lysates and associated with virus-like particles VLPs collected from TCS. Treatment with rocaglamide, but not with ouabain, caused a reduction in both LCMV and LASV Z budding efficiency Fig 6C and 6D . The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments.",
"The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments. The first experiment involved the use of a newly generated single-cycle infectious recombinant LCMV expressing the reporter gene ZsGreen scrLCMV/ZsG-P2A-NP to infect MOI = 0.1 A549 cells 1 st infection . These cells were subsequently transfected with a plasmid expressing LCMV GPC. After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F .",
"After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F . The second experiment involved infection MOI = 0.1 of cells with WT LCMV, and 48 h later we washed infected cells three times to remove the extracellular infectious progeny produced during the first 48 h of infection. Then, fresh media containing ouabain or DMSO vehicle control were added, and 24 h later we determined virus titers in TCS Fig 6G . Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections.",
"Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections. Since ouabain and rocaglamide inhibited different steps of the LCMV life cycle, we examined whether their use in combination results in a synergistic anti-LCMV effect. For this experiment, we infected A549 cells with rLCMV/eGFP and treated them with ouabain and rocaglamide using different concentrations and combinations. At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide.",
"At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide. We next asked whether the ATP1A1 and PHB host-cell factors contributed also to multiplication of viral hemorrhagic fever-causing LASV. We treated A549 cells with ouabain, bufalin, or rocaglamide and inoculated the treated cells with recombinant LASV expressing eGFP rLASV/eGFP . eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 .",
"eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 . We also tested the effect of compounds targeting ATP1A1 and PHB on multiplication of JUNV. Consistent with our results obtained with LCMV and LASV, ouabain, bufalin, and rocaglamide strongly suppressed JUNV multiplication S3 Fig . These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses.",
"These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses. Our approach using a recombinant LCMV expressing NP with an affinity tag facilitated defining the NP interactome in the context of LCMV infection. Recently, using an NP-specific monoclonal antibody mAb to precipitate NP and associated cellular protein partners in a mammarenavirus NP interactome, King et al. identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al.",
"identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al. Differences in both experimental conditions and analysis methodologies used to generate the LCMV NP interactome documented by King et al. and ours likely h post-transfection, cells were washed with fresh media to eliminate Z-mediated production of VLPs in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain or Roc-A at the indicated concentrations. VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D .",
"VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D . Budding efficiency for each sample was estimated by dividing the signal intensity of the Z protein associated with VLPs by that of Z detected in the cell lysate. Numbers on the bottom of panel C correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments.",
"Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments. F Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected MOI = 0.1 with scrLCMV/ZsG 1 st infection for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared.",
"At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. ZsGreen signal intensity was measured by a fluorescent plate reader. GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%.",
"GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%. Data represent means ± SD from three independent experiments. contributed to the observed differences between data sets. Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems.",
"Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems. We used results of the eGFP-Strep interactome, determined in r3LCMV/eGFP-Strep-infected cells, as a control to filter out non-specific NP interactions, which may have resulted in a higher degree of stringency than in the study by King et al for selection of NP-interacting candidates. The combined information provided by the NP interactome reported by King et al. and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction .",
"and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction . The anti-IFN activity of NP was mapped to its C-terminal part and linked to the 3'-5' exonuclease domain present with the NP C-terminal part . Inhibitor-B kinase ε IKKε was identified as an NP-binding protein using plasmid-mediated overexpression in transfected cells , and the NP-IKKε binding affinity correlated with NP's ability to inhibit IFN-I induction . We, as well as the work by King et al. , did not detect this NP-IKKε interaction.",
"We, as well as the work by King et al. , did not detect this NP-IKKε interaction. This discrepancy may have been caused by very low expression of IKKε in LCMV-infected cells, which prevented detection of IKKε by LC-MS/MS. Alternatively, NP-IKKε interaction could possibility be temporarily regulated and take place at early times pi, but could be mostly absent at 48 h pi, the time at which we prepared the cell lysates for our proteomics studies. Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit .",
"Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit . ATP1A1 represents one of four α subunits . Recent evidence has suggested that the Na + /K + -ATPase is involved in multiple cell signaling pathways that are independent of its ion-pumping function . Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase .",
"Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase . Thus, a low concentration of ouabain induces a conformational change in ATP1A1 that results in activation and release of proto-oncogene tyrosine protein kinase, Src, from ATP1A1, followed by activation of as yet unknown downstream signaling that inhibits, for instance, cell entry of murine hepatitis virus MHV . However, our results indicated that ouabain did not interfere with LCMV cell entry. In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV.",
"In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV. Moreover, cell entry of JUNV occurs also by clathrin-mediated endocytosis , a process affected by Src signaling. Ouabain has been clinically used in several European countries for the management of congestive heart failure, whereas bufalin has been tested in clinical trials for cancer treatments , and the CST digoxin has been FDA-approved since 1997 to treat heart failure and atrial fibrillation. Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry.",
"Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry. In contrast, PHB was reported to be a cell entry receptor for DENV-2 and CHIKV . On the other hand, PHB did not act as a virus cell entry receptor for HCV. Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV .",
"Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV . These findings indicate that PHB is involved in different steps of the life cycle of a variety of viruses, and thereby an attractive target for the development of broad-spectrum antiviral drugs. Rocaglate is a group of natural compounds, which includes rocaglamide, that inhibits protein synthesis by targeting the ATP-dependent DEAD-box RNA helicase eukaryotic initiation factor 4A eIF4A and exerts anti-tumor activity . The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication.",
"The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication. These proteins, which included MAP1B, might have anti-LCMV activity. MAP1B has been shown to bind to nonstructural proteins 1 NS1 and 2 NS2 of human respiratory syncytial virus HRSV . NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined .",
"NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined . Examining the role of NP-MAP1B interaction in modulating NP's ability to inhibit induction of type I IFN is of interest. We identified among the NP-interacting host cell proteins the RNA helicase Moloney leukemia virus 10 MOV10 , which has been reported to be an antiviral factor for FLUAV , retroviruses , and DENV-2 . We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions.",
"We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions. MOV10 was shown to enhance IRF-3-mediated IFN-I induction following SeV infection through a tank binding kinase 1 TBK1 -independent and IKKε-dependent manner. This finding was further supported by demonstrating MOV10-IKKε interaction by co-immunoprecipitation studies . We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined.",
"We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined. Several members of the mammalian chaperonin-containing T-complex CCT were identified as prominent hits in our NP interactome. The mammalian CCT is critical for folding of many proteins with important functions in diverse cellular processes , and may protect complex protein topologies within its central cavity during biosynthesis and folding . The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV .",
"The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV . However, the role of these CCT proteins in virus multiplication remains unknown and may involve functions other than acting as molecular chaperones. Previous studies documented the presence of several components of the of eIF4F, including 4A, within viral replication-transcription complexes RTC detected in cells infected with LCMV and TCRV . These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity.",
"These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity. In this work, we documented the generation of rLCMV/Strep-NP and its use to define the NP-interactome in infected cells. We presented evidence that ATP1A1 and PHB contribute to efficient multiplication of mammarenaviruses using genetics and pharmacological inhibition of the genes. Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins.",
"Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins. Future studies elucidating the roles of pro-and antiviral host-cell factors identified in this study in mammarenavirus multiplication will advance our understanding of the multiple functions of NP and uncover novel cellular targets for the development of antimammarenaviral drugs. In addition, by identifying proviral host-cell factors, drugs that are already approved can be repurposing as therapeutics to combat human pathogenic mammarenavirus infections. Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described .",
"Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described . Generation of rLCMV/NP D382A and SeV, strain Cantell, was described 30, 82, 83 . An rLCMV lacking GPC and expressing eGFP rLCMVΔGPC/eGFP was generated by reverse genetics using procedures previously described . rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B .",
"rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B . The rLCMV expressing eGFP rLCMV/eGFP was generated as described , and the rLCMV expressing ZsGreen rLCMV/ZsG instead of eGFP was generated by reverse genetics using similar procedures to generate rLCMV/eGFP. Generation of rLASV expressing eGFP rLASV/eGFP will be described elsewhere. A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG.",
"A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG. scrLCMV/ZsG-P2A-NP was rescued by reverse genetics using similar procedures to generate rLCMVΔGPC/eGFP . LCMV titers were determined by immunofocus forming assay IFFA as described . Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA .",
"Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA . VSV titers were determined by a plaque assay. Total cell lysates were prepared in PD lysis buffer + 250 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% TritonX-100, 10% glycerol, 1 mM of MgCl 2 , 1 μM of CaCl 2 , 1 μM of ZnCl 2 and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA .",
"Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA . To detect Strep-tagged proteins, membranes were reacted with mouse monoclonal antibodies to Strep QIAGEN, Germantown, MD , eGFP Takara Bio USA, Mountain View, CA , GP 2 We33/ 36 , ATP1A1 TehrmoFisher Scientific, Rockford, IL , PHB Abcam, Cambridge, MA or rabbit polyclonal antibodies to α-tubulin Cell Signaling Technologies, Danvers, MA or glyceraldehyde-3-phosphate dehydrogenase GAPDH, Millipore , respectively, followed by incubation with appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G IgG antibodies Jackson ImmunoResearch Laboratories, West Grove, PA . SuperSignal West Pico or Femto chemiluminescent substrate Thermo Fisher Scientific was used to elicit chemiluminescent signals that were visualized using ImageQuant LAS 4000 Imager GE Healthcare Bio-Sciences, Pittsburgh, PA . Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1.",
"Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1. At 48 h pi, cells were washed three times with ice-cold PBS, scraped into fresh ice-cold PBS, and centrifuged at 400 x g at 4˚C for 10 min. Supernatant was removed, and cells were lysed with 12 ml of PD lysis buffer + supplemented with halt protease and phosphatase inhibitor cocktail Thermo Fisher Scientific and 5 μg/ml of deoxyribonuclease I Worthington Biochemical Corporation, Lakewood, NJ . Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C.",
"Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C. After 2 h of incubation, the resin was washed three times with PD lysis buffer + and once with PD lysis buffer without TritonX-100 PD lysis buffer - . After the centrifugation at 1,600 x g and 4˚C for 5 min, the last wash buffer was removed, and protein complexes associated with the resin were eluted into 2 ml of PD lysis buffer - containing 2.5 mM of desthiobiotin. The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn.",
"The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn. A MudPIT microcolumn was prepared by first creating a Kasil frit at one end of an un-deactivated 250-μm outside diameter OD capillary tube interior diameter of 360 μm Agilent Technologies, Inc., Santa Clara, CA . The Kasil frit was prepared by briefly dipping a 20-30-cm capillary tube in 300 μl of Kasil 1624 potassium silicate well-mixed solution PQ Corporation, Malvern, PA and 100 μl of formamide, curing at 100˚C for 4 h, and cutting the frit to a length of %2 mm. Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading.",
"Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading. MudPIT analysis. An analytical reversed-phase liquid chromatography column was generated by pulling a 100-μm interior diameter ID of 360 μm OD capillary tube Polymicro Technologies, Phoenix, AZ to 5-μm ID tip. Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid .",
"Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid . MudPIT and analytical columns were assembled using a zero-dead volume union Upchurch Scientific, Oak Harbor, WA . LC-MS/MS analysis was performed with an Agilent high-pressure LC pump Agilent and linear quadrupole ion dual cell trap Orbitrap Velos Thermo using an in-house built electrospray stage. Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient.",
"Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient. Data analysis. Protein and peptide identification were performed with Integrated Proteomics Pipeline-IP2 Integrated Proteomics Applications, San Diego, CA. integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in.",
"integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in. Spectrum raw files were extracted into ms2 files from raw files using open source RawExtract 1.9.9 Scripps Research Institute, La Jolla, CA; and the tandem mass spectra were searched against a human protein database UniprotKB . To accurately estimate peptide probabilities and false discovery rates, we used a decoy database containing the reversed sequences of all the proteins appended to the target database. Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system.",
"Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system. The search space included half and fully tryptic peptide candidates that fell within the mass tolerance window with no miscleavage constraint. Carbamidomethylation +57.02146 Da of cysteine was considered as a static modification. siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG.",
"siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG. siRNA target host-cell proteins were selected based on availability of validated siRNA sequences. The siRNAs we used to examine the effects on LCMV multiplication of knockdown expression of NP-interacting host cell protein candidate hits corresponded to the Genome-wide ON TAR-GET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene; Dharmacon, Lafayette, CO . A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min.",
"A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. The total protein concentration of clarified cell lysate was measured by Pierce BCA Protein Assay Kit Thermo Fisher Scientific . The same amount of protein from each sample was subjected to SDS-PAGE, and the protein expression of siRNA-targeted genes was analyzed by western blots. Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI .",
"Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI . Green fluorescence eGFP or ZsGreen and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, Bio-Tek, Winooski, VT . Mock-and virus-infected cells were fixed with 4% PFA. After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488.",
"After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488. In some samples, primary antibody against ATP1A1 or PHB was omitted to determine background fluorescence. To visualize nuclei, DAPI Fluoromount-G SouthernBiotech, Birmingham, AL was used to mount coverslips on a slide glass. Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss .",
"Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss . Eight green NP-positive cells were marked and every pixel in the marked area was plotted in the scatter diagram based on its intensity level from each channel. Thresholds for green and red channels were determined using mock-infected cells stained with VL-4-AF488 anti-NP and anti-mouse IgG antibody conjugated with Alexa Fluor 568, without using anti-ATP1A1 or -PHB antibodies. Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels.",
"Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels. This calculation was repeated for eight individual cells. To assess the specificity of co-localization, we determined weighted CC by taking into consideration the brightness of each channel signal. Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software.",
"Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software. A549 or Vero E6 cells seeded 2.0 x 10 4 cells/well in a 96-well plate and cultured overnight were treated with 3-fold serial compound dilutions at 37˚C and 5% CO 2 for 2 h, followed by infection with rLCMV/eGFP MOI = 0.01 . Compounds were present to study endpoint. At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%.",
"At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%. The IC 50 concentrations were determined using GraphPad Prism. A549 or Vero E6 cells seeded in a 96-well plate 2.0 x 10 4 cells/well and cultured overnight were treated with 3-fold serial compound dilutions and cultured at 37˚C and 5% CO 2 for 48 h. Then, CellTiter 96 AQ ueous one solution reagent Promega, Madison, WI was added. Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%.",
"Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%. The CC 50 concentrations were determined using GraphPad Prism. A plasmid expressing C-terminus Strep-tagged Z protein pC-LCMV-Z-Strep was generated using similar procedure to generate a plasmid expressing C-terminus FLAG-tagged LASV Z protein pC-LASV-Z-FLAG , and the budding assay was performed as previously described . Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium.",
"Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium. After the removal of the last wash medium, cells were cultured in fresh medium containing ouabain 30 or 40 nM or rocaglamide 50 or 100 nM or equivalent concentration of DMSO, and 24 h later, virion-like particle VLP -containing TCS and cells were collected. Total cell lysate was prepared by lysing the cells with lysis buffer 1% NP-40, 50 mM of Tris-HCl pH 8.0 , 62.5 mM NaCl, 0.4% sodium deoxycholate . After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots.",
"After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots. A549 cells infected with rLCMV/eGFP were harvested using Accutase cell detachment solution Innovative Cell Technologies, San Diego, CA and fixed with 4% PFA in PBS. eGFP expression was examined by flow cytometry using a BD LSR II Becton Dickson , and data were analyzed with FlowJo Tree Star, Inc., Ashland, OR . 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO .",
"293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO . At 48 h pi, TCS was collected and used to infect fresh monolayers of BHK-21 cells seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . A549 cells seeded 2 x 10 5 cells/well in a 96-well plate and cultured overnight were treated with combinations of different concentrations of ouabain and rocaglamide for 2 h and then infected MOI = 0.01 with rLCMV/eGFP. Compounds were present in the culture medium throughout the experiment. At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . eGFP readouts were normalized by DAPI readouts, and normalized data were used to analyze synergistic effect of the two compounds by the MacSynergy II program . Data were analyzed for p values by a two-tailed unpaired t test using GraphPad Prism software."
] | 1,687 | 5,156 |
What method can significantly alleviate the emergence of drug-resistant variants in RNA viral infections? | Combination therapy | [
"Several mammalian arenaviruses mammarenaviruses cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus LCMV , is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors.",
"Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.",
"We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. Text: Introduction Mammarenaviruses Arenaviridae: Mammarenavirus cause chronic infections of rodents worldwide . Invasion of human dwellings by infected rodents can result in human infections through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW .",
"Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW . The OW Lassa virus LASV , causative agent of Lassa fever LF , is the most significant OW mammarenaviral pathogen. LASV is estimated to infect several hundred thousand individuals annually in Western Africa, resulting in a high number of LF cases associated with high morbidity and lethality. Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever .",
"Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever . The worldwide-distributed OW mammarenavirus lymphocytic choriomeningitis virus LCMV is a neglected human pathogen of clinical significance especially in congenital infections . Moreover, LCMV poses a particular threat to immunocompromised individuals, as has been illustrated by fatal cases of LCMV infection associated with organ transplants . However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina.",
"However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina. Likewise, current anti-mammarenavirus therapy is limited to an offlabel use of the nucleoside analogue ribavirin that is only partially effective and can cause significant side effects . Development of effective anti-mammarenavirus drugs has been hampered by the lack of detailed understanding of virus/host-cell interactions required for mammarenavirus multiplication that could represent amenable targets for antiviral therapy. the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B .",
"the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B . To facilitate the identification of specific PPI between NP and host cell proteins, we used our mammarenavirus tri-segmented r3 platform to design an r3LCMV expressing a C-terminus Strep-tag version of enhanced green fluorescent protein r3LCMV/eGFP-Strep that we used as a negative control Fig 1A and 1B . We rescued rLCMV/Strep-NP and r3LCMV/eGFP-Strep and confirmed the expression of strep-tagged NP and eGFP in rLCMV/ Strep-NP-and r3LCMV/eGFP-Strep-infected cells, respectively Fig 1C . Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV.",
"Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV. However, both rLCMV/Strep-NP and r3LCMV/eGFP-Strep had WT-like growth kinetics and reached high titers. As with WT LCMV, infection with rLCMV/Strep-NP prevented production of bioactive IFN-I by cells in response to Sendai virus SeV infection as determined using an IFN bioassay based on protection against the cytopathic effect CPE induced by infection with vesicular stomatitis virus VSV Fig 1E . Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE.",
"Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE. We selected the human A549 cell line because lung epithelial cells are one of the initial cell targets of humans following inhalation of mammarenavirions. We infected A549 cells multiplicity of infection MOI = 0.1 with either rLCMV/Strep-NP or r3LCMV/eGFP-Strep Fig 2A . At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining.",
"At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining. We compared the pattern of stained protein bands detected between rLCMV/Strep-NP-and r3LCMV/eGFP-Strepinfected samples and confirmed the presence of Strep-NP and eGFP-Strep in pertinent samples Fig 2B . Protein complexes in the rest of eluates from the PD samples were concentrated by trichloroacetic acid TCA precipitation and subjected to trypsin digestion Fig 2A . Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 .",
"Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 . Filtering the data using these criteria resulted in identification of 139 candidate host-cell proteins as NP-interacting partners. Among 53 proteins found present in both NP-and eGFP-PD samples, 36 had spectral counts in the NP-PD sample that were less than two-fold higher than their corresponding spectral counts in the eGFP-PD sample Fig 2C and S1 Table . Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV.",
"Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV. The protein annotation through evolutionary relationship PANTHER protein family classification Biological Process of the NP-interacting protein candidates revealed that a large number of proteins were involved in metabolic and cellular processes Fig 2D . We also analyzed the molecular functions of the NP-interacting protein candidates according to the PAN-THER protein profile classification Fig 2E , which revealed diversified biochemical functions enriched for nucleic acid-binding proteins and chaperones. To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene .",
"To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene . OTPs are latest generation of siRNAs and offer significant advantages over previous generations. Off-target effects are primarily driven by antisense strand microRNA miR -like seed activity. In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy.",
"In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy. Numerous host-cell factors showed an anti-LCMV effect increased ZsG expression by kd of the genes , including microtubule-associated protein 1B MAP1B , dengue virus 2 DENV-2 , and chikungunya virus CHIKV . To begin assessing the potential biological implications of the identified NP-host cell protein interactions, we selected ATP1A1 and PHB given the availability of reagents, existing knowledge about their roles in cell physiology, and evidence of participation in multiplication of other viruses. We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers.",
"We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers. For this, we transfected A549 cells with siRNA targeting Expression of Strep-tagged proteins. A549 cells seeded 5.0 x 10 5 cells/well in a 6-well plate and cultured overnight were infected MOI = 0.1 with the indicated rLCMVs. At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins.",
"At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins. BHK-21 1.75 x 10 5 cells/well , A549 1.25 x 10 5 cells/well , or Vero E6 1.25 x 10 5 cells/well cells seeded in 24-well plates and cultured overnight were infected MOI = 0.01 with the indicated rLCMVs. At the indicated times pi, TCSs were collected and virus titers determined by IFFA. Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP.",
"Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP. A549 cells were infected MOI = 0.1 with the indicated rLCMV or mock-infected, and 36 h later infected with SeV MOI = 1 . At 24 h pi with SeV, TCS were collected and used, following virus inactivation by U.V., to treat Vero E6 cells for 16 h, followed by infection with rVSV MOI = 0.1 rVSV + or mockinfection rVSV - . At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production.",
"At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production. ATP1A1 or with NC siRNA 72 h prior to infection with rLCMV/ZsG. We found that siRNAmediated kd of ATP1A1 dramatically inhibited ZsGreen expression Fig 3Ci , which was associated with a significant reduction of infectious LCMV progeny Fig 3Cii . Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both.",
"Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both. Another consideration is that only a minor fraction of NP might be engaged in the interaction with a given host cell protein, and therefore, detection of these interactions would require highly sensitive methods such as LC-MS/MS. To overcome this problem we used confocal microscopy to examine the co-localization of NP with ATP1A1 and PHB in LCMV-infected cells. Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries .",
"Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries . The PHB inhibitor rocaglamide is a flavagline from an Aglaia tree used in traditional Chinese medicine that has potent anticancer activity . To examine whether pharmacological inhibition of ATP1A1 or PHB inhibited LCMV multiplication, we pretreated human A549 and HEK 293T , nonhuman primate Vero E6 , and rodent murine L929 and hamster BHK-21 cells with ouabain or rocaglamide and infected them with rLCMV/eGFP S1 Fig . Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition .",
"Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition . Likewise, we observed a dose-dependent rocaglamide inhibition of eGFP expression in all cell lines infected with rLCMV/eGFP S1B Fig . Consistent with these findings, production of infectious LCMV progeny was reduced by treatment with either ouabain or rocaglamide Fig 5A within a concentration range that had minimal impact on cell viability Fig 5B . To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability.",
"To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability. To gain insights about the mechanisms by which ouabain and rocaglamide exert their anti-LCMV activity, we examined effects of these compounds on distinct steps of the LCMV life cycle. First, we asked whether ouabain and rocaglamide affected cell entry of LCMV. We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection.",
"We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection. The timing of compound addition did not significantly change the number of eGFP-positive cells, indicating that neither ouabain nor rocaglamide inhibited cell entry of LCMV. The number of eGFP + cells in ouabain-treated cells was reduced at all time-of-addition points compared to vehicle dimethyl sulfoxide DMSO -treated cells, but was similar to that observed in ammonium chloride-treated cells. Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription.",
"Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription. To further investigate the effect of ouabain and rocaglamide on virus RNA synthesis, we infected A549 cells with a recombinant single-cycle infectious LCMV expressing eGFP rLCMVΔGPC/eGFP and treated cells with either ouabain or rocaglamide. Seventy-two hours later, we determined percent normalized eGFP expression in infected cells Fig 6B . Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis.",
"Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis. We also examined the effect of ouabain and rocaglamide on the late step of the arenavirus life cycle, Z-mediated budding. For this experiment, we transfected cells with Z-Strep-and Z-FLAG DYKDDDDK epitope -expressing plasmids from LCMV and LASV, respectively. At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide.",
"At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide. At 24 h, we determined by WB levels of Z protein in both whole cell lysates and associated with virus-like particles VLPs collected from TCS. Treatment with rocaglamide, but not with ouabain, caused a reduction in both LCMV and LASV Z budding efficiency Fig 6C and 6D . The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments.",
"The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments. The first experiment involved the use of a newly generated single-cycle infectious recombinant LCMV expressing the reporter gene ZsGreen scrLCMV/ZsG-P2A-NP to infect MOI = 0.1 A549 cells 1 st infection . These cells were subsequently transfected with a plasmid expressing LCMV GPC. After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F .",
"After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F . The second experiment involved infection MOI = 0.1 of cells with WT LCMV, and 48 h later we washed infected cells three times to remove the extracellular infectious progeny produced during the first 48 h of infection. Then, fresh media containing ouabain or DMSO vehicle control were added, and 24 h later we determined virus titers in TCS Fig 6G . Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections.",
"Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections. Since ouabain and rocaglamide inhibited different steps of the LCMV life cycle, we examined whether their use in combination results in a synergistic anti-LCMV effect. For this experiment, we infected A549 cells with rLCMV/eGFP and treated them with ouabain and rocaglamide using different concentrations and combinations. At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide.",
"At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide. We next asked whether the ATP1A1 and PHB host-cell factors contributed also to multiplication of viral hemorrhagic fever-causing LASV. We treated A549 cells with ouabain, bufalin, or rocaglamide and inoculated the treated cells with recombinant LASV expressing eGFP rLASV/eGFP . eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 .",
"eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 . We also tested the effect of compounds targeting ATP1A1 and PHB on multiplication of JUNV. Consistent with our results obtained with LCMV and LASV, ouabain, bufalin, and rocaglamide strongly suppressed JUNV multiplication S3 Fig . These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses.",
"These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses. Our approach using a recombinant LCMV expressing NP with an affinity tag facilitated defining the NP interactome in the context of LCMV infection. Recently, using an NP-specific monoclonal antibody mAb to precipitate NP and associated cellular protein partners in a mammarenavirus NP interactome, King et al. identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al.",
"identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al. Differences in both experimental conditions and analysis methodologies used to generate the LCMV NP interactome documented by King et al. and ours likely h post-transfection, cells were washed with fresh media to eliminate Z-mediated production of VLPs in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain or Roc-A at the indicated concentrations. VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D .",
"VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D . Budding efficiency for each sample was estimated by dividing the signal intensity of the Z protein associated with VLPs by that of Z detected in the cell lysate. Numbers on the bottom of panel C correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments.",
"Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments. F Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected MOI = 0.1 with scrLCMV/ZsG 1 st infection for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared.",
"At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. ZsGreen signal intensity was measured by a fluorescent plate reader. GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%.",
"GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%. Data represent means ± SD from three independent experiments. contributed to the observed differences between data sets. Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems.",
"Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems. We used results of the eGFP-Strep interactome, determined in r3LCMV/eGFP-Strep-infected cells, as a control to filter out non-specific NP interactions, which may have resulted in a higher degree of stringency than in the study by King et al for selection of NP-interacting candidates. The combined information provided by the NP interactome reported by King et al. and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction .",
"and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction . The anti-IFN activity of NP was mapped to its C-terminal part and linked to the 3'-5' exonuclease domain present with the NP C-terminal part . Inhibitor-B kinase ε IKKε was identified as an NP-binding protein using plasmid-mediated overexpression in transfected cells , and the NP-IKKε binding affinity correlated with NP's ability to inhibit IFN-I induction . We, as well as the work by King et al. , did not detect this NP-IKKε interaction.",
"We, as well as the work by King et al. , did not detect this NP-IKKε interaction. This discrepancy may have been caused by very low expression of IKKε in LCMV-infected cells, which prevented detection of IKKε by LC-MS/MS. Alternatively, NP-IKKε interaction could possibility be temporarily regulated and take place at early times pi, but could be mostly absent at 48 h pi, the time at which we prepared the cell lysates for our proteomics studies. Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit .",
"Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit . ATP1A1 represents one of four α subunits . Recent evidence has suggested that the Na + /K + -ATPase is involved in multiple cell signaling pathways that are independent of its ion-pumping function . Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase .",
"Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase . Thus, a low concentration of ouabain induces a conformational change in ATP1A1 that results in activation and release of proto-oncogene tyrosine protein kinase, Src, from ATP1A1, followed by activation of as yet unknown downstream signaling that inhibits, for instance, cell entry of murine hepatitis virus MHV . However, our results indicated that ouabain did not interfere with LCMV cell entry. In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV.",
"In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV. Moreover, cell entry of JUNV occurs also by clathrin-mediated endocytosis , a process affected by Src signaling. Ouabain has been clinically used in several European countries for the management of congestive heart failure, whereas bufalin has been tested in clinical trials for cancer treatments , and the CST digoxin has been FDA-approved since 1997 to treat heart failure and atrial fibrillation. Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry.",
"Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry. In contrast, PHB was reported to be a cell entry receptor for DENV-2 and CHIKV . On the other hand, PHB did not act as a virus cell entry receptor for HCV. Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV .",
"Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV . These findings indicate that PHB is involved in different steps of the life cycle of a variety of viruses, and thereby an attractive target for the development of broad-spectrum antiviral drugs. Rocaglate is a group of natural compounds, which includes rocaglamide, that inhibits protein synthesis by targeting the ATP-dependent DEAD-box RNA helicase eukaryotic initiation factor 4A eIF4A and exerts anti-tumor activity . The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication.",
"The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication. These proteins, which included MAP1B, might have anti-LCMV activity. MAP1B has been shown to bind to nonstructural proteins 1 NS1 and 2 NS2 of human respiratory syncytial virus HRSV . NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined .",
"NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined . Examining the role of NP-MAP1B interaction in modulating NP's ability to inhibit induction of type I IFN is of interest. We identified among the NP-interacting host cell proteins the RNA helicase Moloney leukemia virus 10 MOV10 , which has been reported to be an antiviral factor for FLUAV , retroviruses , and DENV-2 . We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions.",
"We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions. MOV10 was shown to enhance IRF-3-mediated IFN-I induction following SeV infection through a tank binding kinase 1 TBK1 -independent and IKKε-dependent manner. This finding was further supported by demonstrating MOV10-IKKε interaction by co-immunoprecipitation studies . We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined.",
"We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined. Several members of the mammalian chaperonin-containing T-complex CCT were identified as prominent hits in our NP interactome. The mammalian CCT is critical for folding of many proteins with important functions in diverse cellular processes , and may protect complex protein topologies within its central cavity during biosynthesis and folding . The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV .",
"The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV . However, the role of these CCT proteins in virus multiplication remains unknown and may involve functions other than acting as molecular chaperones. Previous studies documented the presence of several components of the of eIF4F, including 4A, within viral replication-transcription complexes RTC detected in cells infected with LCMV and TCRV . These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity.",
"These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity. In this work, we documented the generation of rLCMV/Strep-NP and its use to define the NP-interactome in infected cells. We presented evidence that ATP1A1 and PHB contribute to efficient multiplication of mammarenaviruses using genetics and pharmacological inhibition of the genes. Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins.",
"Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins. Future studies elucidating the roles of pro-and antiviral host-cell factors identified in this study in mammarenavirus multiplication will advance our understanding of the multiple functions of NP and uncover novel cellular targets for the development of antimammarenaviral drugs. In addition, by identifying proviral host-cell factors, drugs that are already approved can be repurposing as therapeutics to combat human pathogenic mammarenavirus infections. Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described .",
"Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described . Generation of rLCMV/NP D382A and SeV, strain Cantell, was described 30, 82, 83 . An rLCMV lacking GPC and expressing eGFP rLCMVΔGPC/eGFP was generated by reverse genetics using procedures previously described . rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B .",
"rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B . The rLCMV expressing eGFP rLCMV/eGFP was generated as described , and the rLCMV expressing ZsGreen rLCMV/ZsG instead of eGFP was generated by reverse genetics using similar procedures to generate rLCMV/eGFP. Generation of rLASV expressing eGFP rLASV/eGFP will be described elsewhere. A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG.",
"A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG. scrLCMV/ZsG-P2A-NP was rescued by reverse genetics using similar procedures to generate rLCMVΔGPC/eGFP . LCMV titers were determined by immunofocus forming assay IFFA as described . Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA .",
"Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA . VSV titers were determined by a plaque assay. Total cell lysates were prepared in PD lysis buffer + 250 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% TritonX-100, 10% glycerol, 1 mM of MgCl 2 , 1 μM of CaCl 2 , 1 μM of ZnCl 2 and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA .",
"Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA . To detect Strep-tagged proteins, membranes were reacted with mouse monoclonal antibodies to Strep QIAGEN, Germantown, MD , eGFP Takara Bio USA, Mountain View, CA , GP 2 We33/ 36 , ATP1A1 TehrmoFisher Scientific, Rockford, IL , PHB Abcam, Cambridge, MA or rabbit polyclonal antibodies to α-tubulin Cell Signaling Technologies, Danvers, MA or glyceraldehyde-3-phosphate dehydrogenase GAPDH, Millipore , respectively, followed by incubation with appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G IgG antibodies Jackson ImmunoResearch Laboratories, West Grove, PA . SuperSignal West Pico or Femto chemiluminescent substrate Thermo Fisher Scientific was used to elicit chemiluminescent signals that were visualized using ImageQuant LAS 4000 Imager GE Healthcare Bio-Sciences, Pittsburgh, PA . Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1.",
"Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1. At 48 h pi, cells were washed three times with ice-cold PBS, scraped into fresh ice-cold PBS, and centrifuged at 400 x g at 4˚C for 10 min. Supernatant was removed, and cells were lysed with 12 ml of PD lysis buffer + supplemented with halt protease and phosphatase inhibitor cocktail Thermo Fisher Scientific and 5 μg/ml of deoxyribonuclease I Worthington Biochemical Corporation, Lakewood, NJ . Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C.",
"Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C. After 2 h of incubation, the resin was washed three times with PD lysis buffer + and once with PD lysis buffer without TritonX-100 PD lysis buffer - . After the centrifugation at 1,600 x g and 4˚C for 5 min, the last wash buffer was removed, and protein complexes associated with the resin were eluted into 2 ml of PD lysis buffer - containing 2.5 mM of desthiobiotin. The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn.",
"The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn. A MudPIT microcolumn was prepared by first creating a Kasil frit at one end of an un-deactivated 250-μm outside diameter OD capillary tube interior diameter of 360 μm Agilent Technologies, Inc., Santa Clara, CA . The Kasil frit was prepared by briefly dipping a 20-30-cm capillary tube in 300 μl of Kasil 1624 potassium silicate well-mixed solution PQ Corporation, Malvern, PA and 100 μl of formamide, curing at 100˚C for 4 h, and cutting the frit to a length of %2 mm. Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading.",
"Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading. MudPIT analysis. An analytical reversed-phase liquid chromatography column was generated by pulling a 100-μm interior diameter ID of 360 μm OD capillary tube Polymicro Technologies, Phoenix, AZ to 5-μm ID tip. Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid .",
"Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid . MudPIT and analytical columns were assembled using a zero-dead volume union Upchurch Scientific, Oak Harbor, WA . LC-MS/MS analysis was performed with an Agilent high-pressure LC pump Agilent and linear quadrupole ion dual cell trap Orbitrap Velos Thermo using an in-house built electrospray stage. Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient.",
"Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient. Data analysis. Protein and peptide identification were performed with Integrated Proteomics Pipeline-IP2 Integrated Proteomics Applications, San Diego, CA. integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in.",
"integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in. Spectrum raw files were extracted into ms2 files from raw files using open source RawExtract 1.9.9 Scripps Research Institute, La Jolla, CA; and the tandem mass spectra were searched against a human protein database UniprotKB . To accurately estimate peptide probabilities and false discovery rates, we used a decoy database containing the reversed sequences of all the proteins appended to the target database. Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system.",
"Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system. The search space included half and fully tryptic peptide candidates that fell within the mass tolerance window with no miscleavage constraint. Carbamidomethylation +57.02146 Da of cysteine was considered as a static modification. siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG.",
"siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG. siRNA target host-cell proteins were selected based on availability of validated siRNA sequences. The siRNAs we used to examine the effects on LCMV multiplication of knockdown expression of NP-interacting host cell protein candidate hits corresponded to the Genome-wide ON TAR-GET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene; Dharmacon, Lafayette, CO . A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min.",
"A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. The total protein concentration of clarified cell lysate was measured by Pierce BCA Protein Assay Kit Thermo Fisher Scientific . The same amount of protein from each sample was subjected to SDS-PAGE, and the protein expression of siRNA-targeted genes was analyzed by western blots. Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI .",
"Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI . Green fluorescence eGFP or ZsGreen and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, Bio-Tek, Winooski, VT . Mock-and virus-infected cells were fixed with 4% PFA. After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488.",
"After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488. In some samples, primary antibody against ATP1A1 or PHB was omitted to determine background fluorescence. To visualize nuclei, DAPI Fluoromount-G SouthernBiotech, Birmingham, AL was used to mount coverslips on a slide glass. Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss .",
"Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss . Eight green NP-positive cells were marked and every pixel in the marked area was plotted in the scatter diagram based on its intensity level from each channel. Thresholds for green and red channels were determined using mock-infected cells stained with VL-4-AF488 anti-NP and anti-mouse IgG antibody conjugated with Alexa Fluor 568, without using anti-ATP1A1 or -PHB antibodies. Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels.",
"Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels. This calculation was repeated for eight individual cells. To assess the specificity of co-localization, we determined weighted CC by taking into consideration the brightness of each channel signal. Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software.",
"Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software. A549 or Vero E6 cells seeded 2.0 x 10 4 cells/well in a 96-well plate and cultured overnight were treated with 3-fold serial compound dilutions at 37˚C and 5% CO 2 for 2 h, followed by infection with rLCMV/eGFP MOI = 0.01 . Compounds were present to study endpoint. At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%.",
"At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%. The IC 50 concentrations were determined using GraphPad Prism. A549 or Vero E6 cells seeded in a 96-well plate 2.0 x 10 4 cells/well and cultured overnight were treated with 3-fold serial compound dilutions and cultured at 37˚C and 5% CO 2 for 48 h. Then, CellTiter 96 AQ ueous one solution reagent Promega, Madison, WI was added. Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%.",
"Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%. The CC 50 concentrations were determined using GraphPad Prism. A plasmid expressing C-terminus Strep-tagged Z protein pC-LCMV-Z-Strep was generated using similar procedure to generate a plasmid expressing C-terminus FLAG-tagged LASV Z protein pC-LASV-Z-FLAG , and the budding assay was performed as previously described . Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium.",
"Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium. After the removal of the last wash medium, cells were cultured in fresh medium containing ouabain 30 or 40 nM or rocaglamide 50 or 100 nM or equivalent concentration of DMSO, and 24 h later, virion-like particle VLP -containing TCS and cells were collected. Total cell lysate was prepared by lysing the cells with lysis buffer 1% NP-40, 50 mM of Tris-HCl pH 8.0 , 62.5 mM NaCl, 0.4% sodium deoxycholate . After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots.",
"After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots. A549 cells infected with rLCMV/eGFP were harvested using Accutase cell detachment solution Innovative Cell Technologies, San Diego, CA and fixed with 4% PFA in PBS. eGFP expression was examined by flow cytometry using a BD LSR II Becton Dickson , and data were analyzed with FlowJo Tree Star, Inc., Ashland, OR . 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO .",
"293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO . At 48 h pi, TCS was collected and used to infect fresh monolayers of BHK-21 cells seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . A549 cells seeded 2 x 10 5 cells/well in a 96-well plate and cultured overnight were treated with combinations of different concentrations of ouabain and rocaglamide for 2 h and then infected MOI = 0.01 with rLCMV/eGFP. Compounds were present in the culture medium throughout the experiment. At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . eGFP readouts were normalized by DAPI readouts, and normalized data were used to analyze synergistic effect of the two compounds by the MacSynergy II program . Data were analyzed for p values by a two-tailed unpaired t test using GraphPad Prism software."
] | 1,687 | 5,157 |
What factors did this study attribute to the efficient multiplication of mammarenaviruses? | ATP1A1 and PHB | [
"Several mammalian arenaviruses mammarenaviruses cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus LCMV , is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors.",
"Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.",
"We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. Text: Introduction Mammarenaviruses Arenaviridae: Mammarenavirus cause chronic infections of rodents worldwide . Invasion of human dwellings by infected rodents can result in human infections through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW .",
"Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW . The OW Lassa virus LASV , causative agent of Lassa fever LF , is the most significant OW mammarenaviral pathogen. LASV is estimated to infect several hundred thousand individuals annually in Western Africa, resulting in a high number of LF cases associated with high morbidity and lethality. Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever .",
"Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever . The worldwide-distributed OW mammarenavirus lymphocytic choriomeningitis virus LCMV is a neglected human pathogen of clinical significance especially in congenital infections . Moreover, LCMV poses a particular threat to immunocompromised individuals, as has been illustrated by fatal cases of LCMV infection associated with organ transplants . However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina.",
"However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina. Likewise, current anti-mammarenavirus therapy is limited to an offlabel use of the nucleoside analogue ribavirin that is only partially effective and can cause significant side effects . Development of effective anti-mammarenavirus drugs has been hampered by the lack of detailed understanding of virus/host-cell interactions required for mammarenavirus multiplication that could represent amenable targets for antiviral therapy. the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B .",
"the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B . To facilitate the identification of specific PPI between NP and host cell proteins, we used our mammarenavirus tri-segmented r3 platform to design an r3LCMV expressing a C-terminus Strep-tag version of enhanced green fluorescent protein r3LCMV/eGFP-Strep that we used as a negative control Fig 1A and 1B . We rescued rLCMV/Strep-NP and r3LCMV/eGFP-Strep and confirmed the expression of strep-tagged NP and eGFP in rLCMV/ Strep-NP-and r3LCMV/eGFP-Strep-infected cells, respectively Fig 1C . Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV.",
"Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV. However, both rLCMV/Strep-NP and r3LCMV/eGFP-Strep had WT-like growth kinetics and reached high titers. As with WT LCMV, infection with rLCMV/Strep-NP prevented production of bioactive IFN-I by cells in response to Sendai virus SeV infection as determined using an IFN bioassay based on protection against the cytopathic effect CPE induced by infection with vesicular stomatitis virus VSV Fig 1E . Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE.",
"Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE. We selected the human A549 cell line because lung epithelial cells are one of the initial cell targets of humans following inhalation of mammarenavirions. We infected A549 cells multiplicity of infection MOI = 0.1 with either rLCMV/Strep-NP or r3LCMV/eGFP-Strep Fig 2A . At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining.",
"At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining. We compared the pattern of stained protein bands detected between rLCMV/Strep-NP-and r3LCMV/eGFP-Strepinfected samples and confirmed the presence of Strep-NP and eGFP-Strep in pertinent samples Fig 2B . Protein complexes in the rest of eluates from the PD samples were concentrated by trichloroacetic acid TCA precipitation and subjected to trypsin digestion Fig 2A . Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 .",
"Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 . Filtering the data using these criteria resulted in identification of 139 candidate host-cell proteins as NP-interacting partners. Among 53 proteins found present in both NP-and eGFP-PD samples, 36 had spectral counts in the NP-PD sample that were less than two-fold higher than their corresponding spectral counts in the eGFP-PD sample Fig 2C and S1 Table . Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV.",
"Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV. The protein annotation through evolutionary relationship PANTHER protein family classification Biological Process of the NP-interacting protein candidates revealed that a large number of proteins were involved in metabolic and cellular processes Fig 2D . We also analyzed the molecular functions of the NP-interacting protein candidates according to the PAN-THER protein profile classification Fig 2E , which revealed diversified biochemical functions enriched for nucleic acid-binding proteins and chaperones. To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene .",
"To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene . OTPs are latest generation of siRNAs and offer significant advantages over previous generations. Off-target effects are primarily driven by antisense strand microRNA miR -like seed activity. In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy.",
"In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy. Numerous host-cell factors showed an anti-LCMV effect increased ZsG expression by kd of the genes , including microtubule-associated protein 1B MAP1B , dengue virus 2 DENV-2 , and chikungunya virus CHIKV . To begin assessing the potential biological implications of the identified NP-host cell protein interactions, we selected ATP1A1 and PHB given the availability of reagents, existing knowledge about their roles in cell physiology, and evidence of participation in multiplication of other viruses. We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers.",
"We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers. For this, we transfected A549 cells with siRNA targeting Expression of Strep-tagged proteins. A549 cells seeded 5.0 x 10 5 cells/well in a 6-well plate and cultured overnight were infected MOI = 0.1 with the indicated rLCMVs. At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins.",
"At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins. BHK-21 1.75 x 10 5 cells/well , A549 1.25 x 10 5 cells/well , or Vero E6 1.25 x 10 5 cells/well cells seeded in 24-well plates and cultured overnight were infected MOI = 0.01 with the indicated rLCMVs. At the indicated times pi, TCSs were collected and virus titers determined by IFFA. Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP.",
"Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP. A549 cells were infected MOI = 0.1 with the indicated rLCMV or mock-infected, and 36 h later infected with SeV MOI = 1 . At 24 h pi with SeV, TCS were collected and used, following virus inactivation by U.V., to treat Vero E6 cells for 16 h, followed by infection with rVSV MOI = 0.1 rVSV + or mockinfection rVSV - . At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production.",
"At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production. ATP1A1 or with NC siRNA 72 h prior to infection with rLCMV/ZsG. We found that siRNAmediated kd of ATP1A1 dramatically inhibited ZsGreen expression Fig 3Ci , which was associated with a significant reduction of infectious LCMV progeny Fig 3Cii . Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both.",
"Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both. Another consideration is that only a minor fraction of NP might be engaged in the interaction with a given host cell protein, and therefore, detection of these interactions would require highly sensitive methods such as LC-MS/MS. To overcome this problem we used confocal microscopy to examine the co-localization of NP with ATP1A1 and PHB in LCMV-infected cells. Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries .",
"Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries . The PHB inhibitor rocaglamide is a flavagline from an Aglaia tree used in traditional Chinese medicine that has potent anticancer activity . To examine whether pharmacological inhibition of ATP1A1 or PHB inhibited LCMV multiplication, we pretreated human A549 and HEK 293T , nonhuman primate Vero E6 , and rodent murine L929 and hamster BHK-21 cells with ouabain or rocaglamide and infected them with rLCMV/eGFP S1 Fig . Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition .",
"Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition . Likewise, we observed a dose-dependent rocaglamide inhibition of eGFP expression in all cell lines infected with rLCMV/eGFP S1B Fig . Consistent with these findings, production of infectious LCMV progeny was reduced by treatment with either ouabain or rocaglamide Fig 5A within a concentration range that had minimal impact on cell viability Fig 5B . To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability.",
"To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability. To gain insights about the mechanisms by which ouabain and rocaglamide exert their anti-LCMV activity, we examined effects of these compounds on distinct steps of the LCMV life cycle. First, we asked whether ouabain and rocaglamide affected cell entry of LCMV. We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection.",
"We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection. The timing of compound addition did not significantly change the number of eGFP-positive cells, indicating that neither ouabain nor rocaglamide inhibited cell entry of LCMV. The number of eGFP + cells in ouabain-treated cells was reduced at all time-of-addition points compared to vehicle dimethyl sulfoxide DMSO -treated cells, but was similar to that observed in ammonium chloride-treated cells. Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription.",
"Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription. To further investigate the effect of ouabain and rocaglamide on virus RNA synthesis, we infected A549 cells with a recombinant single-cycle infectious LCMV expressing eGFP rLCMVΔGPC/eGFP and treated cells with either ouabain or rocaglamide. Seventy-two hours later, we determined percent normalized eGFP expression in infected cells Fig 6B . Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis.",
"Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis. We also examined the effect of ouabain and rocaglamide on the late step of the arenavirus life cycle, Z-mediated budding. For this experiment, we transfected cells with Z-Strep-and Z-FLAG DYKDDDDK epitope -expressing plasmids from LCMV and LASV, respectively. At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide.",
"At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide. At 24 h, we determined by WB levels of Z protein in both whole cell lysates and associated with virus-like particles VLPs collected from TCS. Treatment with rocaglamide, but not with ouabain, caused a reduction in both LCMV and LASV Z budding efficiency Fig 6C and 6D . The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments.",
"The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments. The first experiment involved the use of a newly generated single-cycle infectious recombinant LCMV expressing the reporter gene ZsGreen scrLCMV/ZsG-P2A-NP to infect MOI = 0.1 A549 cells 1 st infection . These cells were subsequently transfected with a plasmid expressing LCMV GPC. After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F .",
"After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F . The second experiment involved infection MOI = 0.1 of cells with WT LCMV, and 48 h later we washed infected cells three times to remove the extracellular infectious progeny produced during the first 48 h of infection. Then, fresh media containing ouabain or DMSO vehicle control were added, and 24 h later we determined virus titers in TCS Fig 6G . Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections.",
"Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections. Since ouabain and rocaglamide inhibited different steps of the LCMV life cycle, we examined whether their use in combination results in a synergistic anti-LCMV effect. For this experiment, we infected A549 cells with rLCMV/eGFP and treated them with ouabain and rocaglamide using different concentrations and combinations. At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide.",
"At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide. We next asked whether the ATP1A1 and PHB host-cell factors contributed also to multiplication of viral hemorrhagic fever-causing LASV. We treated A549 cells with ouabain, bufalin, or rocaglamide and inoculated the treated cells with recombinant LASV expressing eGFP rLASV/eGFP . eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 .",
"eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 . We also tested the effect of compounds targeting ATP1A1 and PHB on multiplication of JUNV. Consistent with our results obtained with LCMV and LASV, ouabain, bufalin, and rocaglamide strongly suppressed JUNV multiplication S3 Fig . These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses.",
"These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses. Our approach using a recombinant LCMV expressing NP with an affinity tag facilitated defining the NP interactome in the context of LCMV infection. Recently, using an NP-specific monoclonal antibody mAb to precipitate NP and associated cellular protein partners in a mammarenavirus NP interactome, King et al. identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al.",
"identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al. Differences in both experimental conditions and analysis methodologies used to generate the LCMV NP interactome documented by King et al. and ours likely h post-transfection, cells were washed with fresh media to eliminate Z-mediated production of VLPs in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain or Roc-A at the indicated concentrations. VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D .",
"VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D . Budding efficiency for each sample was estimated by dividing the signal intensity of the Z protein associated with VLPs by that of Z detected in the cell lysate. Numbers on the bottom of panel C correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments.",
"Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments. F Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected MOI = 0.1 with scrLCMV/ZsG 1 st infection for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared.",
"At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. ZsGreen signal intensity was measured by a fluorescent plate reader. GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%.",
"GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%. Data represent means ± SD from three independent experiments. contributed to the observed differences between data sets. Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems.",
"Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems. We used results of the eGFP-Strep interactome, determined in r3LCMV/eGFP-Strep-infected cells, as a control to filter out non-specific NP interactions, which may have resulted in a higher degree of stringency than in the study by King et al for selection of NP-interacting candidates. The combined information provided by the NP interactome reported by King et al. and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction .",
"and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction . The anti-IFN activity of NP was mapped to its C-terminal part and linked to the 3'-5' exonuclease domain present with the NP C-terminal part . Inhibitor-B kinase ε IKKε was identified as an NP-binding protein using plasmid-mediated overexpression in transfected cells , and the NP-IKKε binding affinity correlated with NP's ability to inhibit IFN-I induction . We, as well as the work by King et al. , did not detect this NP-IKKε interaction.",
"We, as well as the work by King et al. , did not detect this NP-IKKε interaction. This discrepancy may have been caused by very low expression of IKKε in LCMV-infected cells, which prevented detection of IKKε by LC-MS/MS. Alternatively, NP-IKKε interaction could possibility be temporarily regulated and take place at early times pi, but could be mostly absent at 48 h pi, the time at which we prepared the cell lysates for our proteomics studies. Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit .",
"Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit . ATP1A1 represents one of four α subunits . Recent evidence has suggested that the Na + /K + -ATPase is involved in multiple cell signaling pathways that are independent of its ion-pumping function . Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase .",
"Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase . Thus, a low concentration of ouabain induces a conformational change in ATP1A1 that results in activation and release of proto-oncogene tyrosine protein kinase, Src, from ATP1A1, followed by activation of as yet unknown downstream signaling that inhibits, for instance, cell entry of murine hepatitis virus MHV . However, our results indicated that ouabain did not interfere with LCMV cell entry. In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV.",
"In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV. Moreover, cell entry of JUNV occurs also by clathrin-mediated endocytosis , a process affected by Src signaling. Ouabain has been clinically used in several European countries for the management of congestive heart failure, whereas bufalin has been tested in clinical trials for cancer treatments , and the CST digoxin has been FDA-approved since 1997 to treat heart failure and atrial fibrillation. Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry.",
"Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry. In contrast, PHB was reported to be a cell entry receptor for DENV-2 and CHIKV . On the other hand, PHB did not act as a virus cell entry receptor for HCV. Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV .",
"Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV . These findings indicate that PHB is involved in different steps of the life cycle of a variety of viruses, and thereby an attractive target for the development of broad-spectrum antiviral drugs. Rocaglate is a group of natural compounds, which includes rocaglamide, that inhibits protein synthesis by targeting the ATP-dependent DEAD-box RNA helicase eukaryotic initiation factor 4A eIF4A and exerts anti-tumor activity . The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication.",
"The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication. These proteins, which included MAP1B, might have anti-LCMV activity. MAP1B has been shown to bind to nonstructural proteins 1 NS1 and 2 NS2 of human respiratory syncytial virus HRSV . NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined .",
"NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined . Examining the role of NP-MAP1B interaction in modulating NP's ability to inhibit induction of type I IFN is of interest. We identified among the NP-interacting host cell proteins the RNA helicase Moloney leukemia virus 10 MOV10 , which has been reported to be an antiviral factor for FLUAV , retroviruses , and DENV-2 . We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions.",
"We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions. MOV10 was shown to enhance IRF-3-mediated IFN-I induction following SeV infection through a tank binding kinase 1 TBK1 -independent and IKKε-dependent manner. This finding was further supported by demonstrating MOV10-IKKε interaction by co-immunoprecipitation studies . We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined.",
"We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined. Several members of the mammalian chaperonin-containing T-complex CCT were identified as prominent hits in our NP interactome. The mammalian CCT is critical for folding of many proteins with important functions in diverse cellular processes , and may protect complex protein topologies within its central cavity during biosynthesis and folding . The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV .",
"The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV . However, the role of these CCT proteins in virus multiplication remains unknown and may involve functions other than acting as molecular chaperones. Previous studies documented the presence of several components of the of eIF4F, including 4A, within viral replication-transcription complexes RTC detected in cells infected with LCMV and TCRV . These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity.",
"These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity. In this work, we documented the generation of rLCMV/Strep-NP and its use to define the NP-interactome in infected cells. We presented evidence that ATP1A1 and PHB contribute to efficient multiplication of mammarenaviruses using genetics and pharmacological inhibition of the genes. Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins.",
"Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins. Future studies elucidating the roles of pro-and antiviral host-cell factors identified in this study in mammarenavirus multiplication will advance our understanding of the multiple functions of NP and uncover novel cellular targets for the development of antimammarenaviral drugs. In addition, by identifying proviral host-cell factors, drugs that are already approved can be repurposing as therapeutics to combat human pathogenic mammarenavirus infections. Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described .",
"Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described . Generation of rLCMV/NP D382A and SeV, strain Cantell, was described 30, 82, 83 . An rLCMV lacking GPC and expressing eGFP rLCMVΔGPC/eGFP was generated by reverse genetics using procedures previously described . rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B .",
"rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B . The rLCMV expressing eGFP rLCMV/eGFP was generated as described , and the rLCMV expressing ZsGreen rLCMV/ZsG instead of eGFP was generated by reverse genetics using similar procedures to generate rLCMV/eGFP. Generation of rLASV expressing eGFP rLASV/eGFP will be described elsewhere. A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG.",
"A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG. scrLCMV/ZsG-P2A-NP was rescued by reverse genetics using similar procedures to generate rLCMVΔGPC/eGFP . LCMV titers were determined by immunofocus forming assay IFFA as described . Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA .",
"Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA . VSV titers were determined by a plaque assay. Total cell lysates were prepared in PD lysis buffer + 250 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% TritonX-100, 10% glycerol, 1 mM of MgCl 2 , 1 μM of CaCl 2 , 1 μM of ZnCl 2 and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA .",
"Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA . To detect Strep-tagged proteins, membranes were reacted with mouse monoclonal antibodies to Strep QIAGEN, Germantown, MD , eGFP Takara Bio USA, Mountain View, CA , GP 2 We33/ 36 , ATP1A1 TehrmoFisher Scientific, Rockford, IL , PHB Abcam, Cambridge, MA or rabbit polyclonal antibodies to α-tubulin Cell Signaling Technologies, Danvers, MA or glyceraldehyde-3-phosphate dehydrogenase GAPDH, Millipore , respectively, followed by incubation with appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G IgG antibodies Jackson ImmunoResearch Laboratories, West Grove, PA . SuperSignal West Pico or Femto chemiluminescent substrate Thermo Fisher Scientific was used to elicit chemiluminescent signals that were visualized using ImageQuant LAS 4000 Imager GE Healthcare Bio-Sciences, Pittsburgh, PA . Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1.",
"Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1. At 48 h pi, cells were washed three times with ice-cold PBS, scraped into fresh ice-cold PBS, and centrifuged at 400 x g at 4˚C for 10 min. Supernatant was removed, and cells were lysed with 12 ml of PD lysis buffer + supplemented with halt protease and phosphatase inhibitor cocktail Thermo Fisher Scientific and 5 μg/ml of deoxyribonuclease I Worthington Biochemical Corporation, Lakewood, NJ . Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C.",
"Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C. After 2 h of incubation, the resin was washed three times with PD lysis buffer + and once with PD lysis buffer without TritonX-100 PD lysis buffer - . After the centrifugation at 1,600 x g and 4˚C for 5 min, the last wash buffer was removed, and protein complexes associated with the resin were eluted into 2 ml of PD lysis buffer - containing 2.5 mM of desthiobiotin. The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn.",
"The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn. A MudPIT microcolumn was prepared by first creating a Kasil frit at one end of an un-deactivated 250-μm outside diameter OD capillary tube interior diameter of 360 μm Agilent Technologies, Inc., Santa Clara, CA . The Kasil frit was prepared by briefly dipping a 20-30-cm capillary tube in 300 μl of Kasil 1624 potassium silicate well-mixed solution PQ Corporation, Malvern, PA and 100 μl of formamide, curing at 100˚C for 4 h, and cutting the frit to a length of %2 mm. Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading.",
"Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading. MudPIT analysis. An analytical reversed-phase liquid chromatography column was generated by pulling a 100-μm interior diameter ID of 360 μm OD capillary tube Polymicro Technologies, Phoenix, AZ to 5-μm ID tip. Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid .",
"Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid . MudPIT and analytical columns were assembled using a zero-dead volume union Upchurch Scientific, Oak Harbor, WA . LC-MS/MS analysis was performed with an Agilent high-pressure LC pump Agilent and linear quadrupole ion dual cell trap Orbitrap Velos Thermo using an in-house built electrospray stage. Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient.",
"Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient. Data analysis. Protein and peptide identification were performed with Integrated Proteomics Pipeline-IP2 Integrated Proteomics Applications, San Diego, CA. integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in.",
"integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in. Spectrum raw files were extracted into ms2 files from raw files using open source RawExtract 1.9.9 Scripps Research Institute, La Jolla, CA; and the tandem mass spectra were searched against a human protein database UniprotKB . To accurately estimate peptide probabilities and false discovery rates, we used a decoy database containing the reversed sequences of all the proteins appended to the target database. Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system.",
"Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system. The search space included half and fully tryptic peptide candidates that fell within the mass tolerance window with no miscleavage constraint. Carbamidomethylation +57.02146 Da of cysteine was considered as a static modification. siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG.",
"siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG. siRNA target host-cell proteins were selected based on availability of validated siRNA sequences. The siRNAs we used to examine the effects on LCMV multiplication of knockdown expression of NP-interacting host cell protein candidate hits corresponded to the Genome-wide ON TAR-GET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene; Dharmacon, Lafayette, CO . A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min.",
"A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. The total protein concentration of clarified cell lysate was measured by Pierce BCA Protein Assay Kit Thermo Fisher Scientific . The same amount of protein from each sample was subjected to SDS-PAGE, and the protein expression of siRNA-targeted genes was analyzed by western blots. Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI .",
"Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI . Green fluorescence eGFP or ZsGreen and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, Bio-Tek, Winooski, VT . Mock-and virus-infected cells were fixed with 4% PFA. After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488.",
"After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488. In some samples, primary antibody against ATP1A1 or PHB was omitted to determine background fluorescence. To visualize nuclei, DAPI Fluoromount-G SouthernBiotech, Birmingham, AL was used to mount coverslips on a slide glass. Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss .",
"Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss . Eight green NP-positive cells were marked and every pixel in the marked area was plotted in the scatter diagram based on its intensity level from each channel. Thresholds for green and red channels were determined using mock-infected cells stained with VL-4-AF488 anti-NP and anti-mouse IgG antibody conjugated with Alexa Fluor 568, without using anti-ATP1A1 or -PHB antibodies. Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels.",
"Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels. This calculation was repeated for eight individual cells. To assess the specificity of co-localization, we determined weighted CC by taking into consideration the brightness of each channel signal. Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software.",
"Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software. A549 or Vero E6 cells seeded 2.0 x 10 4 cells/well in a 96-well plate and cultured overnight were treated with 3-fold serial compound dilutions at 37˚C and 5% CO 2 for 2 h, followed by infection with rLCMV/eGFP MOI = 0.01 . Compounds were present to study endpoint. At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%.",
"At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%. The IC 50 concentrations were determined using GraphPad Prism. A549 or Vero E6 cells seeded in a 96-well plate 2.0 x 10 4 cells/well and cultured overnight were treated with 3-fold serial compound dilutions and cultured at 37˚C and 5% CO 2 for 48 h. Then, CellTiter 96 AQ ueous one solution reagent Promega, Madison, WI was added. Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%.",
"Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%. The CC 50 concentrations were determined using GraphPad Prism. A plasmid expressing C-terminus Strep-tagged Z protein pC-LCMV-Z-Strep was generated using similar procedure to generate a plasmid expressing C-terminus FLAG-tagged LASV Z protein pC-LASV-Z-FLAG , and the budding assay was performed as previously described . Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium.",
"Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium. After the removal of the last wash medium, cells were cultured in fresh medium containing ouabain 30 or 40 nM or rocaglamide 50 or 100 nM or equivalent concentration of DMSO, and 24 h later, virion-like particle VLP -containing TCS and cells were collected. Total cell lysate was prepared by lysing the cells with lysis buffer 1% NP-40, 50 mM of Tris-HCl pH 8.0 , 62.5 mM NaCl, 0.4% sodium deoxycholate . After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots.",
"After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots. A549 cells infected with rLCMV/eGFP were harvested using Accutase cell detachment solution Innovative Cell Technologies, San Diego, CA and fixed with 4% PFA in PBS. eGFP expression was examined by flow cytometry using a BD LSR II Becton Dickson , and data were analyzed with FlowJo Tree Star, Inc., Ashland, OR . 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO .",
"293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO . At 48 h pi, TCS was collected and used to infect fresh monolayers of BHK-21 cells seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . A549 cells seeded 2 x 10 5 cells/well in a 96-well plate and cultured overnight were treated with combinations of different concentrations of ouabain and rocaglamide for 2 h and then infected MOI = 0.01 with rLCMV/eGFP. Compounds were present in the culture medium throughout the experiment. At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . eGFP readouts were normalized by DAPI readouts, and normalized data were used to analyze synergistic effect of the two compounds by the MacSynergy II program . Data were analyzed for p values by a two-tailed unpaired t test using GraphPad Prism software."
] | 1,687 | 5,158 |
How do the authors suggest that ATP1A1 and PHB contribute to the efficient multiplication of mammarenaviruses? | using genetics and pharmacological inhibition of the genes | [
"Several mammalian arenaviruses mammarenaviruses cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus LCMV , is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors.",
"Here we document the generation of a recombinant LCMV encoding a nucleoprotein NP containing an affinity tag rLCMV/Strep-NP and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na + /K + transporting subunit alpha 1 ATP1A1 and prohibitin PHB as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans.",
"We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. Text: Introduction Mammarenaviruses Arenaviridae: Mammarenavirus cause chronic infections of rodents worldwide . Invasion of human dwellings by infected rodents can result in human infections through mucosal exposure to aerosols or by direct contact of abraded skin with infectious material. Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW .",
"Several mammarenaviruses cause viral hemorrhagic fevers VHFs in humans and pose important public health problems in their endemic areas . Mammarenaviruses are classified into two main groups, Old World OW and New World NW . The OW Lassa virus LASV , causative agent of Lassa fever LF , is the most significant OW mammarenaviral pathogen. LASV is estimated to infect several hundred thousand individuals annually in Western Africa, resulting in a high number of LF cases associated with high morbidity and lethality. Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever .",
"Moreover, LASV endemic regions are expanding , and the association of the recently identified mammarenavirus Lujo virus with a VHF outbreak in Southern Africa has raised concerns about the emergence of novel VHF-causing mammarenaviruses. The most significant NW mammarenavirus is Junín virus JUNV , which causes Argentinian hemorrhagic fever . The worldwide-distributed OW mammarenavirus lymphocytic choriomeningitis virus LCMV is a neglected human pathogen of clinical significance especially in congenital infections . Moreover, LCMV poses a particular threat to immunocompromised individuals, as has been illustrated by fatal cases of LCMV infection associated with organ transplants . However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina.",
"However, LCMV research can be safely performed at BSL-2 containment, rather than the BSL-4 containment necessary for live LASV or JUNV research . No US Food and Drug Administration FDA -licensed vaccines are available for the treatment of arenavirus infections, although a live attenuated vaccine strain of JUNV, Candid#1, is licensed in Argentina. Likewise, current anti-mammarenavirus therapy is limited to an offlabel use of the nucleoside analogue ribavirin that is only partially effective and can cause significant side effects . Development of effective anti-mammarenavirus drugs has been hampered by the lack of detailed understanding of virus/host-cell interactions required for mammarenavirus multiplication that could represent amenable targets for antiviral therapy. the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B .",
"the potential problem that overexpression of a single viral gene product may potentiate PPI interactions that are not relevant during the course of a natural virus infection. To overcome this issue, we designed a recombinant LCMV rLCMV encoding a tandem WSHPQFEK GGGS 3 WSHPQFEK Strep-tag fused to the amino-terminus of NP rLCMV/Strep-NP Fig 1A and 1B . To facilitate the identification of specific PPI between NP and host cell proteins, we used our mammarenavirus tri-segmented r3 platform to design an r3LCMV expressing a C-terminus Strep-tag version of enhanced green fluorescent protein r3LCMV/eGFP-Strep that we used as a negative control Fig 1A and 1B . We rescued rLCMV/Strep-NP and r3LCMV/eGFP-Strep and confirmed the expression of strep-tagged NP and eGFP in rLCMV/ Strep-NP-and r3LCMV/eGFP-Strep-infected cells, respectively Fig 1C . Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV.",
"Next, we examined the growth properties of rLCMV/Strep-NP and r3LCMV/eGFP-Strep in three different cells lines from hamsters, humans, and nonhuman primates BHK-21, A549, and Vero E6 cells, respectively Fig 1D . The fitness of rLCMV/Strep-NP and r3LCMV/eGFP-Strep was modestly decreased compared to that observed with wild-type WT Armstrong rLCMV ARM and Clone 13 rLCMV Cl-13 strains of LCMV. However, both rLCMV/Strep-NP and r3LCMV/eGFP-Strep had WT-like growth kinetics and reached high titers. As with WT LCMV, infection with rLCMV/Strep-NP prevented production of bioactive IFN-I by cells in response to Sendai virus SeV infection as determined using an IFN bioassay based on protection against the cytopathic effect CPE induced by infection with vesicular stomatitis virus VSV Fig 1E . Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE.",
"Vero cells treated for 16 h with tissue cultured supernatants TCS from A549 cells infected first with WT LCMV or rLCMV/Strep, followed by 24 h infection with SeV, remained fully susceptible to VSV-induced CPE. In contrast, Vero cells treated with TCS from A549 cells infected with rLCMV/NP D382A , a mutant unable to prevent induction of IFN-I , and subsequently with SeV, were protected against VSV induced CPE. We selected the human A549 cell line because lung epithelial cells are one of the initial cell targets of humans following inhalation of mammarenavirions. We infected A549 cells multiplicity of infection MOI = 0.1 with either rLCMV/Strep-NP or r3LCMV/eGFP-Strep Fig 2A . At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining.",
"At 48 h post-inoculation pi , we prepared total cell lysates for pull-down PD assays using a sepharose resin coated with strep-tactin. Aliquots of the protein complexes present in the PD samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE Fig 2B followed by SYPRO Ruby protein gel staining. We compared the pattern of stained protein bands detected between rLCMV/Strep-NP-and r3LCMV/eGFP-Strepinfected samples and confirmed the presence of Strep-NP and eGFP-Strep in pertinent samples Fig 2B . Protein complexes in the rest of eluates from the PD samples were concentrated by trichloroacetic acid TCA precipitation and subjected to trypsin digestion Fig 2A . Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 .",
"Digested peptides were subjected to liquid chromatography-tandem mass spectrometry LC-MS/MS analysis using a hybrid mass spectrometer consisting of linear quadrupole ion dual cell trap LTQ Velos and an Orbitrap analyser. We classified host-cell proteins identified by LC-MS/MS analysis from two independent biological replicates into two groups: 1 proteins found only in Strep-NP PD samples with at least five spectral counts Table 1 , and 2 proteins found in both Strep-NP and eGFP-Strep PD samples with five or higher spectral counts in Strep-NP samples and at least 2-fold higher spectral counts in Strep-NP PD compared to eGFP PD samples Table 2 . Filtering the data using these criteria resulted in identification of 139 candidate host-cell proteins as NP-interacting partners. Among 53 proteins found present in both NP-and eGFP-PD samples, 36 had spectral counts in the NP-PD sample that were less than two-fold higher than their corresponding spectral counts in the eGFP-PD sample Fig 2C and S1 Table . Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV.",
"Based on the criteria we described above, we considered these proteins to be highly unlikely specific NPinteractors with an involvement in the LCMV life cycle, and therefore we did not consider these hits for further analysis. However, we acknowledge that we cannot formally rule out that some of these hits could still play a role on the life cycle of LCMV. The protein annotation through evolutionary relationship PANTHER protein family classification Biological Process of the NP-interacting protein candidates revealed that a large number of proteins were involved in metabolic and cellular processes Fig 2D . We also analyzed the molecular functions of the NP-interacting protein candidates according to the PAN-THER protein profile classification Fig 2E , which revealed diversified biochemical functions enriched for nucleic acid-binding proteins and chaperones. To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene .",
"To initially assess pro-or anti-viral roles of NP-interacting host-cell proteins identified by LC-MS/MS, we examined the effect of small interfering RNA siRNA -mediated knockdown kd of each of the corresponding genes on multiplication of rLCMV expressing reporter gene ZsGreen ZsG in A549 cells Fig 3A . The siRNAs we used were from the genome-wide ON TARGET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene . OTPs are latest generation of siRNAs and offer significant advantages over previous generations. Off-target effects are primarily driven by antisense strand microRNA miR -like seed activity. In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy.",
"In OTPs, the sense strand is modified to favor antisense strand uptake whereas the antisense strand seed region is modified to drastically reduce seed-related off-targeting . In addition, OTPs are designed on the foundation of the SMARTselection algorithm Dharmacon , widely considered to be the best algorithm for rational siRNA design strategy. Numerous host-cell factors showed an anti-LCMV effect increased ZsG expression by kd of the genes , including microtubule-associated protein 1B MAP1B , dengue virus 2 DENV-2 , and chikungunya virus CHIKV . To begin assessing the potential biological implications of the identified NP-host cell protein interactions, we selected ATP1A1 and PHB given the availability of reagents, existing knowledge about their roles in cell physiology, and evidence of participation in multiplication of other viruses. We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers.",
"We confirmed that cells transfected with siRNA specific to ATP1A1 and PHB exhibited the predicted reduced levels in ATP1A1 and PHB protein expression Fig 3B . Likewise, we examined whether siRNA-mediated reduced expression levels of ZsGreen correlated with reduced LCMV progeny titers. For this, we transfected A549 cells with siRNA targeting Expression of Strep-tagged proteins. A549 cells seeded 5.0 x 10 5 cells/well in a 6-well plate and cultured overnight were infected MOI = 0.1 with the indicated rLCMVs. At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins.",
"At 48 h pi, total cell lysates were prepared, and protein expression was analyzed by western blotting. D Growth properties of rLCMV expressing Strep-tagged proteins. BHK-21 1.75 x 10 5 cells/well , A549 1.25 x 10 5 cells/well , or Vero E6 1.25 x 10 5 cells/well cells seeded in 24-well plates and cultured overnight were infected MOI = 0.01 with the indicated rLCMVs. At the indicated times pi, TCSs were collected and virus titers determined by IFFA. Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP.",
"Results represent means ± SD of the results of three independent experiments. E Lack of induction of IFN-I in cells infected with rLCMV/Strep-NP. A549 cells were infected MOI = 0.1 with the indicated rLCMV or mock-infected, and 36 h later infected with SeV MOI = 1 . At 24 h pi with SeV, TCS were collected and used, following virus inactivation by U.V., to treat Vero E6 cells for 16 h, followed by infection with rVSV MOI = 0.1 rVSV + or mockinfection rVSV - . At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production.",
"At 24 h pi with rVSV, cells were fixed with 4% PFA and stained with crystal violet to assess rVSV-induced cytopathic effect. We used as control rLCMV/NP D382A that contains mutation D382A within NP, which has been shown to abrogate the NP's ability to counteract the induction of IFN-I production. ATP1A1 or with NC siRNA 72 h prior to infection with rLCMV/ZsG. We found that siRNAmediated kd of ATP1A1 dramatically inhibited ZsGreen expression Fig 3Ci , which was associated with a significant reduction of infectious LCMV progeny Fig 3Cii . Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both.",
"Our attempts to see interaction between NP and ATP1A1 or NP and PHB by co-immunoprecipitation were unsuccessful. Several possibilities could account for this, including interactions of low affinity or high on/off rate or both. Another consideration is that only a minor fraction of NP might be engaged in the interaction with a given host cell protein, and therefore, detection of these interactions would require highly sensitive methods such as LC-MS/MS. To overcome this problem we used confocal microscopy to examine the co-localization of NP with ATP1A1 and PHB in LCMV-infected cells. Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries .",
"Weighted co-localization coefficients CC , determined by taking into consideration the brightness of each channel signal, were significantly higher than non-weighted CC, indicating the presence of brighter pixels in the co-localized regions compared to the non-co-localized regions Fig 4 . The cardiac glycoside ouabain is an inhibitor of ATP1A1 that has been used to treat congestive heart failure in European countries . The PHB inhibitor rocaglamide is a flavagline from an Aglaia tree used in traditional Chinese medicine that has potent anticancer activity . To examine whether pharmacological inhibition of ATP1A1 or PHB inhibited LCMV multiplication, we pretreated human A549 and HEK 293T , nonhuman primate Vero E6 , and rodent murine L929 and hamster BHK-21 cells with ouabain or rocaglamide and infected them with rLCMV/eGFP S1 Fig . Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition .",
"Ouabain treatment resulted in a strong dosedependent inhibition of eGFP expression in infected human-and nonhuman primate cells, but did not affect eGFP expression intensity in infected rodent cells S1A Fig . This finding is consistent with rodents expressing an ATP1A1 allele that is resistant to ouabain inhibition . Likewise, we observed a dose-dependent rocaglamide inhibition of eGFP expression in all cell lines infected with rLCMV/eGFP S1B Fig . Consistent with these findings, production of infectious LCMV progeny was reduced by treatment with either ouabain or rocaglamide Fig 5A within a concentration range that had minimal impact on cell viability Fig 5B . To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability.",
"To examine the correlation between efficacy and cytotoxicity of these compounds, we determined their therapeutic index TI = CC 50 /IC 50 Fig 5Bi ; whereas rocaglamide had TIs of >105 CC 50 > 1000 nM, IC 50 = 9.51 nM and 10.3 CC 50 = 100 nM, IC 50 = 9.75 nM in A549 and Vero E6 cells, respectively Fig 5Bii . Moreover, the ATP1A1 antagonist inhibitor, bufalin, also exhibited robust anti-LCMV activity with TIs of 8.92 CC 50 Heat shock protein HSP 90-alpha HSP90AA1 P07900 8 10 9 Endoplasmin HSP90B1 P14625 9 9 9 Large neutral amino acids transporter small subunit 1 SLC7A5 Q01650 6 12 9 Keratin, type II cuticular Hb1 KRT81 Q14533 10 8 9 Putative helicase MOV-10 MOV10 Q9HCE1 8 10 9 Microtubule-associated protein 1B MAP1B P46821 6 11 8.5 Spectrin beta chain, rocaglamide 100 nM Fig 5C , further supporting a specific anti-LCMV activity of ouabain and rocaglamide that was not due to reduced cell viability. To gain insights about the mechanisms by which ouabain and rocaglamide exert their anti-LCMV activity, we examined effects of these compounds on distinct steps of the LCMV life cycle. First, we asked whether ouabain and rocaglamide affected cell entry of LCMV. We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection.",
"We conducted time-of-addition experiments in which we treated cells with ouabain or rocaglamide prior to virus inoculation -1.5 h , at the time of inoculation 0 h , or 1.5 h pi +1.5 h Fig 6A . In some samples, we used ammonium chloride starting at 4 h pi to block multiple rounds of virus infection. The timing of compound addition did not significantly change the number of eGFP-positive cells, indicating that neither ouabain nor rocaglamide inhibited cell entry of LCMV. The number of eGFP + cells in ouabain-treated cells was reduced at all time-of-addition points compared to vehicle dimethyl sulfoxide DMSO -treated cells, but was similar to that observed in ammonium chloride-treated cells. Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription.",
"Thus, ouabain did not inhibit LCMV RNA replication and gene expression, but rather a late step of the LCMV life cycle. In contrast, rocaglamide treatment resulted in a negligible number of eGFP + cells, indicating that rocaglamide inhibited virus RNA replication and gene transcription. To further investigate the effect of ouabain and rocaglamide on virus RNA synthesis, we infected A549 cells with a recombinant single-cycle infectious LCMV expressing eGFP rLCMVΔGPC/eGFP and treated cells with either ouabain or rocaglamide. Seventy-two hours later, we determined percent normalized eGFP expression in infected cells Fig 6B . Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis.",
"Consistent with our results from the time-of-addition experiment, ouabain did not affect reporter eGFP expression. However, rocaglamide reduced eGFP expression, confirming inhibitory effect of rocaglamide on virus RNA synthesis. We also examined the effect of ouabain and rocaglamide on the late step of the arenavirus life cycle, Z-mediated budding. For this experiment, we transfected cells with Z-Strep-and Z-FLAG DYKDDDDK epitope -expressing plasmids from LCMV and LASV, respectively. At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide.",
"At 24 h post-transfection, we removed the tissue culture supernatant TCS and washed extensively transfected cells to eliminate already budded Z. We cultured the transfected cells in the presence or absence of ouabain or rocaglamide. At 24 h, we determined by WB levels of Z protein in both whole cell lysates and associated with virus-like particles VLPs collected from TCS. Treatment with rocaglamide, but not with ouabain, caused a reduction in both LCMV and LASV Z budding efficiency Fig 6C and 6D . The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments.",
"The reproducibility of these findings was confirmed based on results from four independent experiments Fig 6E . We also examined whether ouabain could interfere with a step of assembly of infectious progeny that was not captured by the Z budding assay through two additional experiments. The first experiment involved the use of a newly generated single-cycle infectious recombinant LCMV expressing the reporter gene ZsGreen scrLCMV/ZsG-P2A-NP to infect MOI = 0.1 A549 cells 1 st infection . These cells were subsequently transfected with a plasmid expressing LCMV GPC. After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F .",
"After 24 h, we used TCS to infect a fresh cell monolayer 2 nd infection and identified infected cells based on ZsGreen expression. To assess the effect of ouabain on de novo assembly of infectious progeny we determined normalized ratios 2 nd /1 st infection of ZsGreen + cells Fig 6F . The second experiment involved infection MOI = 0.1 of cells with WT LCMV, and 48 h later we washed infected cells three times to remove the extracellular infectious progeny produced during the first 48 h of infection. Then, fresh media containing ouabain or DMSO vehicle control were added, and 24 h later we determined virus titers in TCS Fig 6G . Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections.",
"Results from both experiments consistently showed that ouabain did not inhibit assembly de novo of extracellular infectious virus. Combination therapy can significantly alleviate the problem posed by the rapid emergence of drug-resistant variants commonly observed during monotherapy strategies to control RNA virus infections. Since ouabain and rocaglamide inhibited different steps of the LCMV life cycle, we examined whether their use in combination results in a synergistic anti-LCMV effect. For this experiment, we infected A549 cells with rLCMV/eGFP and treated them with ouabain and rocaglamide using different concentrations and combinations. At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide.",
"At 48 h pi, we determined percent eGFP expression Fig 7 . Combination treatment with ouabain and rocaglamide resulted in synergistic anti-LCMV activity that was enhanced under conditions using higher concentrations of ouabain and lower concentrations of rocaglamide. We next asked whether the ATP1A1 and PHB host-cell factors contributed also to multiplication of viral hemorrhagic fever-causing LASV. We treated A549 cells with ouabain, bufalin, or rocaglamide and inoculated the treated cells with recombinant LASV expressing eGFP rLASV/eGFP . eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 .",
"eGFP expression was examined 48 h later. Similar to rLCMV infection, LASV multiplication was restricted in ouabain-, bufalin-, or rocaglamide-treated cells at concentrations minimally impacting cell viability, although their IC 50 values were slightly higher than those found with the LCMV infection system Fig 5B and S2 Fig as ouabain had IC 50 of 9.34 nM, bufalin had IC 50 of 1.66 nM and rocaglamide had IC 50 of 37.0 nM Fig 8 . We also tested the effect of compounds targeting ATP1A1 and PHB on multiplication of JUNV. Consistent with our results obtained with LCMV and LASV, ouabain, bufalin, and rocaglamide strongly suppressed JUNV multiplication S3 Fig . These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses.",
"These findings indicate that ATP1A1 and PHB function as pro-viral factors of a range of mammarenaviruses. We identified ATP1A1 and PHB as novel host-cell proteins that contribute to efficient multiplication of mammarenaviruses. Our approach using a recombinant LCMV expressing NP with an affinity tag facilitated defining the NP interactome in the context of LCMV infection. Recently, using an NP-specific monoclonal antibody mAb to precipitate NP and associated cellular protein partners in a mammarenavirus NP interactome, King et al. identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al.",
"identified 348 host proteins that associated with LCMV NP . We found 99 common proteins between our filtered LCMV NP interactome of 171 proteins and the LCMV NP interactome documented by King et al. Differences in both experimental conditions and analysis methodologies used to generate the LCMV NP interactome documented by King et al. and ours likely h post-transfection, cells were washed with fresh media to eliminate Z-mediated production of VLPs in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain or Roc-A at the indicated concentrations. VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D .",
"VLPs present in TCS were collected by ultracentrifugation, and cell lysates were prepared. Z protein expression in VLPs and cell lysates were determined by western blots using antibodies to Strep-tag C and FLAG-tag D . Budding efficiency for each sample was estimated by dividing the signal intensity of the Z protein associated with VLPs by that of Z detected in the cell lysate. Numbers on the bottom of panel C correspond to LCMV Z budding efficiencies determined in a representative experiment. Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments.",
"Results shown in panel E correspond to the average and SD from four independent experiments including the one shown in panel D. The mean budding efficiency of DMSO treatedsamples was set to 100%. Data represent mean ± SD of four independent experiments. F Effect of ouabain on incorporation of viral glycoprotein into virions. 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected MOI = 0.1 with scrLCMV/ZsG 1 st infection for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared.",
"At 24 h pi, cells were washed with fresh medium to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of ouabain at 40 nM OUA . At 48 h pi, TCS was collected and used to infect fresh monolayer of BHK-21 cells 2 nd infection seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. ZsGreen signal intensity was measured by a fluorescent plate reader. GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%.",
"GP-incorporation efficiency was estimated by dividing ZsGreen signal intensity in BHK-21 cell lysate 2 nd by that in 293T cell lysate 1 st . The mean GP-incorporation efficiency of DMSO treated samples was set to 100%. Data represent means ± SD from three independent experiments. contributed to the observed differences between data sets. Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems.",
"Despite progress in the implementation of global proteomics-based screens to identify virus-host protein-protein interactions, overlap between datasets for the same viral system is usually limited. However, the substantial overlap of 99 of the 171 NP-interacting proteins from both studies supports the overall reliability of both systems. We used results of the eGFP-Strep interactome, determined in r3LCMV/eGFP-Strep-infected cells, as a control to filter out non-specific NP interactions, which may have resulted in a higher degree of stringency than in the study by King et al for selection of NP-interacting candidates. The combined information provided by the NP interactome reported by King et al. and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction .",
"and the one we present in this work, will facilitate future studies to further characterize the functional and biological implications of NP-host cell interacting proteins. All tested mammarenavirus NPs, with exception of the NP from TCRV, blocked IRF-3-dependent IFN-I induction . The anti-IFN activity of NP was mapped to its C-terminal part and linked to the 3'-5' exonuclease domain present with the NP C-terminal part . Inhibitor-B kinase ε IKKε was identified as an NP-binding protein using plasmid-mediated overexpression in transfected cells , and the NP-IKKε binding affinity correlated with NP's ability to inhibit IFN-I induction . We, as well as the work by King et al. , did not detect this NP-IKKε interaction.",
"We, as well as the work by King et al. , did not detect this NP-IKKε interaction. This discrepancy may have been caused by very low expression of IKKε in LCMV-infected cells, which prevented detection of IKKε by LC-MS/MS. Alternatively, NP-IKKε interaction could possibility be temporarily regulated and take place at early times pi, but could be mostly absent at 48 h pi, the time at which we prepared the cell lysates for our proteomics studies. Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit .",
"Future studies comparing the NP interactome at different times during infection will contribute to a better understanding of the dynamics of NP/hostcell protein interactions. Na + /K + -ATPase is a well-characterized membrane ion transporter and is composed of two functional subunits α and β and one regulatory γ subunit . ATP1A1 represents one of four α subunits . Recent evidence has suggested that the Na + /K + -ATPase is involved in multiple cell signaling pathways that are independent of its ion-pumping function . Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase .",
"Cardiac glycoside inhibitors of the Na + /K + -ATPase NKA , so-called cardiotonic steroids CST; e.g., ouabain, bufalin , have been shown to inhibit multiplication of different viruses including Ebola virus , coronaviruses , herpes simplex virus 1 , CHIKV , human immunodeficiency virus 1 HIV-1 , adenovirus and porcine reproductive and respiratory syndrome virus 1 . Different mechanisms are likely to contribute to the antiviral activity of CSTs, including altered cell functions modulated by the signaling activity of Na + /K + -ATPase . Thus, a low concentration of ouabain induces a conformational change in ATP1A1 that results in activation and release of proto-oncogene tyrosine protein kinase, Src, from ATP1A1, followed by activation of as yet unknown downstream signaling that inhibits, for instance, cell entry of murine hepatitis virus MHV . However, our results indicated that ouabain did not interfere with LCMV cell entry. In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV.",
"In addition, treatment with the Src inhibitor 4-amino-5- 4-methylphenyl -7- t-butyl pyrazolo 3,4-d pyrimidine PP1 did not counteract the anti-LCMV activity of ouabain S4 Fig . Nevertheless, ATP1A1-mediated Src signaling could plausibly contribute to the inhibitory effect of ouabain on JUNV multiplication as similarly to that observed with MHV. Moreover, cell entry of JUNV occurs also by clathrin-mediated endocytosis , a process affected by Src signaling. Ouabain has been clinically used in several European countries for the management of congestive heart failure, whereas bufalin has been tested in clinical trials for cancer treatments , and the CST digoxin has been FDA-approved since 1997 to treat heart failure and atrial fibrillation. Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry.",
"Hence, opportunities for the repurposing CSTs have potential as therapeutics to treat infections caused by viral hemorrhagic fever-causing arenaviruses. The PHB inhibitor, rocaglamide, appeared to interfere with LCMV RNA synthesis and budding, but did not affect LCMV cell entry. In contrast, PHB was reported to be a cell entry receptor for DENV-2 and CHIKV . On the other hand, PHB did not act as a virus cell entry receptor for HCV. Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV .",
"Rather, PHB contributed to HCV cell entry through binding to cellular RAF c-Raf; proto-oncogene serine/threonine-protein kinase and subsequent Harvey rat sarcoma proto-oncogene HRas activation that induces a signal transduction pathway required for epidermal growth factor receptor EGFR -mediated HCV cell entry . In addition, siRNA-mediated kd of PHB decreased production of H5N1 FLUAV . These findings indicate that PHB is involved in different steps of the life cycle of a variety of viruses, and thereby an attractive target for the development of broad-spectrum antiviral drugs. Rocaglate is a group of natural compounds, which includes rocaglamide, that inhibits protein synthesis by targeting the ATP-dependent DEAD-box RNA helicase eukaryotic initiation factor 4A eIF4A and exerts anti-tumor activity . The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication.",
"The rocaglate compound, silvestrol, inhibits Ebola virus multiplication likely by interfering with the role of eIF4A in viral protein translation . While we focused on two host proteins, ATP1A1 and PHB, in this study, our proteomics approach also identified several NP-interacting host-cell proteins whose kd expression via siRNA resulted in increased LCMV multiplication. These proteins, which included MAP1B, might have anti-LCMV activity. MAP1B has been shown to bind to nonstructural proteins 1 NS1 and 2 NS2 of human respiratory syncytial virus HRSV . NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined .",
"NS1 and NS2 of HRSV antagonizes host IFN-I response by reducing the levels of TNF receptor associated factor TRAF3 , IKKε NS1 , and STAT2 NS2 . NS2-MAP1B interaction interfered with HRSV NS2's ability to reduce levels of STAT2, whereas the role of NS1-MAP1B interaction remains to be determined . Examining the role of NP-MAP1B interaction in modulating NP's ability to inhibit induction of type I IFN is of interest. We identified among the NP-interacting host cell proteins the RNA helicase Moloney leukemia virus 10 MOV10 , which has been reported to be an antiviral factor for FLUAV , retroviruses , and DENV-2 . We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions.",
"We did not observe increased LCMV multiplication in cells subjected to siRNA-mediated kd of MOV10, a finding that would question an anti-LCMV activity of MOV10. However, we consider that LCMV has already optimal multiplication in A549 cells and further increases may occur only under rather unique conditions. MOV10 was shown to enhance IRF-3-mediated IFN-I induction following SeV infection through a tank binding kinase 1 TBK1 -independent and IKKε-dependent manner. This finding was further supported by demonstrating MOV10-IKKε interaction by co-immunoprecipitation studies . We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined.",
"We documented that the anti-IFN activity of mammarenavirus NP correlated with its ability to interact with IKKε . Whether NP-MOV10 interaction prevents MOV10 from enhancing IRF-3-mediated IFN-I induction remains to be determined. Several members of the mammalian chaperonin-containing T-complex CCT were identified as prominent hits in our NP interactome. The mammalian CCT is critical for folding of many proteins with important functions in diverse cellular processes , and may protect complex protein topologies within its central cavity during biosynthesis and folding . The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV .",
"The contribution of CCT members to NP assembly into a nucleocapsid structure could account for their presence in the NP, but not eGFP, interactome. Interestingly, members of the CCT have been implicated in multiplication of different viruses including rabies virus , HCV and FLUAV . However, the role of these CCT proteins in virus multiplication remains unknown and may involve functions other than acting as molecular chaperones. Previous studies documented the presence of several components of the of eIF4F, including 4A, within viral replication-transcription complexes RTC detected in cells infected with LCMV and TCRV . These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity.",
"These findings, together with the detection of a number of ribosomal proteins in the NP interactome, suggest that translation of viral mRNAs may take place within RTC. However, rocaglamide interference with the activity of eIF4A within the viral RTC might contribute to its anti-LCMV activity. In this work, we documented the generation of rLCMV/Strep-NP and its use to define the NP-interactome in infected cells. We presented evidence that ATP1A1 and PHB contribute to efficient multiplication of mammarenaviruses using genetics and pharmacological inhibition of the genes. Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins.",
"Consistent with our findings, bioinformatic analysis revealed that the protein network associated with ATP1A1 and PHB involves host cell proteins with functions in biological processes that have been implicated in virus multiplication S5 Fig . The overall experimental approach described here can facilitate the identification of biologically relevant NP-interacting host-cell proteins. Future studies elucidating the roles of pro-and antiviral host-cell factors identified in this study in mammarenavirus multiplication will advance our understanding of the multiple functions of NP and uncover novel cellular targets for the development of antimammarenaviral drugs. In addition, by identifying proviral host-cell factors, drugs that are already approved can be repurposing as therapeutics to combat human pathogenic mammarenavirus infections. Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described .",
"Baby hamster kidney BHK-21 American Type Culture Collection, ATCC, CCL-10 , house mouse L929 ATCC CCL-1 , grivet Vero E6 ATCC CRL-1586 , human A549 ATCC CCL-185 , and human HEK 293T ATCC CRL-3216 cells were grown in Dulbecco's modified Eagle's medium Thermo Fisher Scientific, Waltham, MA containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37˚C and 5% CO 2 . WT recombinant LCMVs, Armstrong rLCMV ARM and clone-13 rLCMV Cl-13 strains, were generated as described . Generation of rLCMV/NP D382A and SeV, strain Cantell, was described 30, 82, 83 . An rLCMV lacking GPC and expressing eGFP rLCMVΔGPC/eGFP was generated by reverse genetics using procedures previously described . rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B .",
"rLCMV/Strep-NP and r3LCMV/eGFP-Strep were generated by reverse genetics using similar procedures to generate WT rLCMV and tri-segmented LCMV r3LCMV expressing eGFP . For the generation of these novel rLCMVs, we created pol1S Cl-13 plasmids that directed Pol1-mediated intracellular synthesis of recombinant LCMV S genome RNA species coding for Strep-tagged NP or eGFP, respectively Fig 1A and 1B . The rLCMV expressing eGFP rLCMV/eGFP was generated as described , and the rLCMV expressing ZsGreen rLCMV/ZsG instead of eGFP was generated by reverse genetics using similar procedures to generate rLCMV/eGFP. Generation of rLASV expressing eGFP rLASV/eGFP will be described elsewhere. A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG.",
"A tri-segmented recombinant live-attenuated Candid #1 strain of JUNV expressing eGFP r3JUNV/eGFP was generated as described . For the generation of a novel single cycle rLCMV expressing ZsGreen scrLCMV/ZsG-P2A-NP , a pol1S plasmid was created by omitting GPC open reading frame ORF from pol1S plasmid used for the generation of rLCMV/ZsG. scrLCMV/ZsG-P2A-NP was rescued by reverse genetics using similar procedures to generate rLCMVΔGPC/eGFP . LCMV titers were determined by immunofocus forming assay IFFA as described . Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA .",
"Briefly, 10-fold serial virus dilutions were used to infect Vero E6 cell monolayers in a 96-well plate, and at 20 h pi, cells were fixed with 4% paraformaldehyde PFA in phosphate-buffered saline PBS . After cell permeabilization by treatment with dilution buffer DB 0.3% Triton X-100 in PBS-containing 3% bovine serum albumin BSA , cells were stained with a rat mAb to NP VL-4, Bio X Cell, West Lebanon, NH conjugated with Alexa Fluor 488 VL-4-AF488, Protein Labeling Kit, Life Technologies, Carlsbad, CA . VSV titers were determined by a plaque assay. Total cell lysates were prepared in PD lysis buffer + 250 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% TritonX-100, 10% glycerol, 1 mM of MgCl 2 , 1 μM of CaCl 2 , 1 μM of ZnCl 2 and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA .",
"Clarified lysates were mixed at a 1:1 ratio with loading buffer 100 mM of Tris pH 6.8 , 20% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol and boiled for 5 min. Proteins samples were fractionated by SDS-PAGE using 4-20% gradient polyacrylamide gels Mini-PROTEAN TGX gels 4-20%, Bio-Rad, Hercules, CA , and proteins were transferred by electroblotting onto polyvinylidene difluoride membranes Immobilin Transfer Membranes, Millipore, Billerica, MA . To detect Strep-tagged proteins, membranes were reacted with mouse monoclonal antibodies to Strep QIAGEN, Germantown, MD , eGFP Takara Bio USA, Mountain View, CA , GP 2 We33/ 36 , ATP1A1 TehrmoFisher Scientific, Rockford, IL , PHB Abcam, Cambridge, MA or rabbit polyclonal antibodies to α-tubulin Cell Signaling Technologies, Danvers, MA or glyceraldehyde-3-phosphate dehydrogenase GAPDH, Millipore , respectively, followed by incubation with appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G IgG antibodies Jackson ImmunoResearch Laboratories, West Grove, PA . SuperSignal West Pico or Femto chemiluminescent substrate Thermo Fisher Scientific was used to elicit chemiluminescent signals that were visualized using ImageQuant LAS 4000 Imager GE Healthcare Bio-Sciences, Pittsburgh, PA . Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1.",
"Pull down of strep-tagged proteins from infected cell lysate. A549 cells prepared in six 15-cm dishes approximately 1.0 x 10 8 cells in total were infected with either rLCMV/Strep-NP or r3LCMV/eGFP at an MOI of 0.1. At 48 h pi, cells were washed three times with ice-cold PBS, scraped into fresh ice-cold PBS, and centrifuged at 400 x g at 4˚C for 10 min. Supernatant was removed, and cells were lysed with 12 ml of PD lysis buffer + supplemented with halt protease and phosphatase inhibitor cocktail Thermo Fisher Scientific and 5 μg/ml of deoxyribonuclease I Worthington Biochemical Corporation, Lakewood, NJ . Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C.",
"Lysate was clarified by centrifugation at 3,900 x g at 4˚C for 30 min to remove cell debris. Clarified cell lysate was then incubated with strep-tactin sepharose resin QIAGEN at 4˚C. After 2 h of incubation, the resin was washed three times with PD lysis buffer + and once with PD lysis buffer without TritonX-100 PD lysis buffer - . After the centrifugation at 1,600 x g and 4˚C for 5 min, the last wash buffer was removed, and protein complexes associated with the resin were eluted into 2 ml of PD lysis buffer - containing 2.5 mM of desthiobiotin. The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn.",
"The eluate was then subjected to TCA precipitation followed by trypsin digestion. Multidimensional protein identification technology microcolumn. A MudPIT microcolumn was prepared by first creating a Kasil frit at one end of an un-deactivated 250-μm outside diameter OD capillary tube interior diameter of 360 μm Agilent Technologies, Inc., Santa Clara, CA . The Kasil frit was prepared by briefly dipping a 20-30-cm capillary tube in 300 μl of Kasil 1624 potassium silicate well-mixed solution PQ Corporation, Malvern, PA and 100 μl of formamide, curing at 100˚C for 4 h, and cutting the frit to a length of %2 mm. Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading.",
"Strong cation exchange particles SCX Luna, 5-μm diameter, 125 Å pores, Phenomenex, Torrance, CA were packed in-house from particle slurries in methanol to 2.5 cm. Reversed phase particles 2 cm, C18 Aqua, 3-μm diameter, 125 Å pores, Phenomenex were then successively packed onto the capillary tube using the same method as SCX loading. MudPIT analysis. An analytical reversed-phase liquid chromatography column was generated by pulling a 100-μm interior diameter ID of 360 μm OD capillary tube Polymicro Technologies, Phoenix, AZ to 5-μm ID tip. Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid .",
"Reversed-phase particles Luna C18, 3-μm diameter, 125 Å pores, Phenomenex were packed directly into the pulled column at 5.5 mPa until 15 cm long. The column was further packed, washed, and equilibrated at 10 mPa with buffer B 80% acetonitrile, 0.1% formic acid followed by buffer A 5% acetonitrile and 0.1% formic acid . MudPIT and analytical columns were assembled using a zero-dead volume union Upchurch Scientific, Oak Harbor, WA . LC-MS/MS analysis was performed with an Agilent high-pressure LC pump Agilent and linear quadrupole ion dual cell trap Orbitrap Velos Thermo using an in-house built electrospray stage. Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient.",
"Electrospray was performed directly from the analytical column by applying the electrospray ionization ESI voltage at a tee 150 μm ID, Upchurch Scientific directly downstream of a 1:1,000 split flow to reduce the flow rate to 300 nl/min through the columns. MudPIT experiments 10-step were performed in which each step corresponds to 0, 10, 20, 40, 50, 60, 70, 80, 90 , and 100% buffer C 500 mM of ammonium acetate, 0.1% formic acid, and 5% acetonitrile and was run for 3 min at the beginning of a 110-min gradient. Data analysis. Protein and peptide identification were performed with Integrated Proteomics Pipeline-IP2 Integrated Proteomics Applications, San Diego, CA. integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in.",
"integratedproteomics.com/ using ProLuCID and DTASelect2 algorithms. DTASelect parameters were-p 2 -y 1-trypstat-pfp .01 -extra-pI-DB-dm-in. Spectrum raw files were extracted into ms2 files from raw files using open source RawExtract 1.9.9 Scripps Research Institute, La Jolla, CA; and the tandem mass spectra were searched against a human protein database UniprotKB . To accurately estimate peptide probabilities and false discovery rates, we used a decoy database containing the reversed sequences of all the proteins appended to the target database. Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system.",
"Tandem mass spectra were matched to sequences using the ProLuCID algorithm with a 600-ppm peptide mass tolerance. ProLuCID searches were done on an Intel Xeon cluster processor running under the Linux operating system. The search space included half and fully tryptic peptide candidates that fell within the mass tolerance window with no miscleavage constraint. Carbamidomethylation +57.02146 Da of cysteine was considered as a static modification. siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG.",
"siRNA screening A549 cells 1,000 cells/well in a 384-well plate were reverse transfected with 0.5 pmol of siRNA pool S2 Table targeting each gene using 0.1 μl of Lipofectamine RNAiMAX Thermo Fisher Scientific final siRNA concentration was 10 nM , followed by incubation at 37˚C and 5% CO 2 . At 72 h post-transfection, cells were infected MOI = 0.05 with rLCMV/ZsG. siRNA target host-cell proteins were selected based on availability of validated siRNA sequences. The siRNAs we used to examine the effects on LCMV multiplication of knockdown expression of NP-interacting host cell protein candidate hits corresponded to the Genome-wide ON TAR-GET-Plus OTP Human siRNA library 18,301 genes, 4 siRNAs/gene; Dharmacon, Lafayette, CO . A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min.",
"A549 cells 3.0 x 10 4 cells/well were reverse transfected in a 24-well plate with 6 pmol of siRNA pools targeting each gene using 1 μl of Lipofectamine RNAiMAX final siRNA concentration is 10 nM . At 72 h post-transfection, total cell lysate was prepared in modified lysis A buffer 25 mM Tris-HCl pH = 8.0 , 50 mM NaCl, 1%Triton X-100, 1.25% sodium deoxycholate and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. The total protein concentration of clarified cell lysate was measured by Pierce BCA Protein Assay Kit Thermo Fisher Scientific . The same amount of protein from each sample was subjected to SDS-PAGE, and the protein expression of siRNA-targeted genes was analyzed by western blots. Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI .",
"Cells infected with eGFP-or ZsGreen-expressing rLCMV were fixed with 4% PFA in PBS. After cell permeabilization by treatment with DB, cells were stained with 4',6-diamidino-2-phenylindole DAPI . Green fluorescence eGFP or ZsGreen and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, Bio-Tek, Winooski, VT . Mock-and virus-infected cells were fixed with 4% PFA. After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488.",
"After cell permeabilization and blocking by treatment with DB containing 1% normal goat serum, cells were incubated with primary mouse anti ATP1A1 or PHB antibody followed by secondary anti-mouse IgG antibody conjugated with Alexa Fluore 568 anti-mouse IgG-AF568 . Subsequently, cells were stained with VL-4-AF488. In some samples, primary antibody against ATP1A1 or PHB was omitted to determine background fluorescence. To visualize nuclei, DAPI Fluoromount-G SouthernBiotech, Birmingham, AL was used to mount coverslips on a slide glass. Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss .",
"Stained cells were observed under a confocal microscope LSM 710, Zeiss and data analyzed by ZEN software Zeiss . Co-localization analysis was performed on a pixel by pixel basis using Zen software Zeiss . Eight green NP-positive cells were marked and every pixel in the marked area was plotted in the scatter diagram based on its intensity level from each channel. Thresholds for green and red channels were determined using mock-infected cells stained with VL-4-AF488 anti-NP and anti-mouse IgG antibody conjugated with Alexa Fluor 568, without using anti-ATP1A1 or -PHB antibodies. Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels.",
"Each pixel was assigned a value of 1. Co-localization coefficients CC or non-weighted CC were determined by dividing the sum of both green-and red-positive pixels by the sum of green positive pixels. This calculation was repeated for eight individual cells. To assess the specificity of co-localization, we determined weighted CC by taking into consideration the brightness of each channel signal. Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software.",
"Comparison of non-weighted and weighted CC allowed us to determine whether brighter pixels were present in the co-localized regions compared to the non-co-localized regions. p values were determined by a two-tailed paired t test using GraphPad Prism software. A549 or Vero E6 cells seeded 2.0 x 10 4 cells/well in a 96-well plate and cultured overnight were treated with 3-fold serial compound dilutions at 37˚C and 5% CO 2 for 2 h, followed by infection with rLCMV/eGFP MOI = 0.01 . Compounds were present to study endpoint. At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%.",
"At 48 h pi, cells were fixed with 4% PFA in PBS, and eGFP expression was examined by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . Mean values obtained with DMSO-treated and rLCMV/eGFP-infected cells were set to 100%. The IC 50 concentrations were determined using GraphPad Prism. A549 or Vero E6 cells seeded in a 96-well plate 2.0 x 10 4 cells/well and cultured overnight were treated with 3-fold serial compound dilutions and cultured at 37˚C and 5% CO 2 for 48 h. Then, CellTiter 96 AQ ueous one solution reagent Promega, Madison, WI was added. Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%.",
"Thereafter, the assay was performed according to the manufacturer's recommendations, and the absorbance 490 nm was obtained using an enzyme-linked immunosorbent assay ELISA reader SPECTRA max plus 384; Molecular Devices, Sunnyvale, CA . Mean values obtained with DMSO-treated cells were set to 100%. The CC 50 concentrations were determined using GraphPad Prism. A plasmid expressing C-terminus Strep-tagged Z protein pC-LCMV-Z-Strep was generated using similar procedure to generate a plasmid expressing C-terminus FLAG-tagged LASV Z protein pC-LASV-Z-FLAG , and the budding assay was performed as previously described . Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium.",
"Cells HEK 293T in a 12-well plate were transfected with 0.5 μg of empty pCAGGS vector or pC-LCMV-Z-Strep or pC-LASV-Z-FLAG using Lipofectamine 2000. At 5 h post-transfection, media were replaced with fresh media and incubated at 37˚C and 5% CO 2 for 19 h. Then the cells were three times washed with fresh medium. After the removal of the last wash medium, cells were cultured in fresh medium containing ouabain 30 or 40 nM or rocaglamide 50 or 100 nM or equivalent concentration of DMSO, and 24 h later, virion-like particle VLP -containing TCS and cells were collected. Total cell lysate was prepared by lysing the cells with lysis buffer 1% NP-40, 50 mM of Tris-HCl pH 8.0 , 62.5 mM NaCl, 0.4% sodium deoxycholate . After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots.",
"After clarification of TCS from cell debris by centrifugation at 400 x g and 4˚C for 10 min, VLPs were collected by ultracentrifugation at 100,000 x g and 4˚C for 30 min through a 20% sucrose cushion. VLPs were resuspended in PBS, and Z expression in total cell lysate and TCS containing VLPs were analyzed by western blots. A549 cells infected with rLCMV/eGFP were harvested using Accutase cell detachment solution Innovative Cell Technologies, San Diego, CA and fixed with 4% PFA in PBS. eGFP expression was examined by flow cytometry using a BD LSR II Becton Dickson , and data were analyzed with FlowJo Tree Star, Inc., Ashland, OR . 293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO .",
"293T cells seeded 4.0 x 10 5 cells/well in a 12-well plate and cultured overnight were infected with scrLCMV/ZsG-P2A-NP for 2 h and subsequently transfected with 0.5 μg of pC-GPC. At 24 h pi, cells were three times washed with fresh media to eliminate infectious virus particle produced in the absence of compound treatment, and cultured for another 24 h in fresh media in the presence of 40 nM of ouabain or vehicle control DMSO . At 48 h pi, TCS was collected and used to infect fresh monolayers of BHK-21 cells seeded 4.0 x 10 5 cells/well in a 12-well plate 1 day before the infection, and 293T cell lysate was prepared. 24 h later, BHK-21 cell lysate was prepared. Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"Total cell lysate was prepared in cell lysis buffer 150 mM of NaCl, 50 mM of Tris-HCl pH = 7.5 , 0.5% NP-40 , 1 mM of EDTA and clarified by centrifugation at 21,130 x g at 4˚C for 10 min. ZsGreen signal intensity in clarified cell lysate was measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . A549 cells seeded 2 x 10 5 cells/well in a 96-well plate and cultured overnight were treated with combinations of different concentrations of ouabain and rocaglamide for 2 h and then infected MOI = 0.01 with rLCMV/eGFP. Compounds were present in the culture medium throughout the experiment. At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek .",
"At 48 h pi, cells were fixed, permeabilized by treatment with DB, and stained with DAPI. eGFP and DAPI signals were measured by a fluorescent plate reader Synergy H4 Hybrid Multi-Mode Microplate Reader, BioTek . eGFP readouts were normalized by DAPI readouts, and normalized data were used to analyze synergistic effect of the two compounds by the MacSynergy II program . Data were analyzed for p values by a two-tailed unpaired t test using GraphPad Prism software."
] | 1,687 | 5,159 |
Which are the most abundant biological entities on Earth? | Viruses | [
"The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution?",
"Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes.",
"Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life.",
"Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.” Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes . . Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids.",
". Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules aptamers , 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds.",
"The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube Lincoln and Joyce, 2009 . Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity.",
"Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid PNA , with more stable peptide bonds instead of phosphodiester bonds Zhang and Cech, 1997; Joyce, 2002 . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides Wilson and Szostak, 1999 . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes.",
"Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs Orgel, 2004 . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids.",
"They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis Meierhenrich, 2008 . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started . . There, temperature gradients and clay containing minerals such as magnesium or manganese are available.",
". There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication . . tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code.",
". tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: \"The ribosome is a ribozyme\" Cech, 2000 , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 .",
"Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 . Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro . . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV . . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . .",
". Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases . . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes Harish and Caetano-Anolles, 2012 . These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase.",
"Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme a reductase as today, and allow for DNA polymerization Wilson and Szostak, 1999; Joyce, 2002 . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin Moelling, 2012 Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . .",
"tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins Moelling and Broecker, 2015; Moelling et al., 2017 Figure 1 . It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the \"Encyclopedia of DNA Elements\" ENCODE project. ncDNA amounts to more than 98% of the human DNA genome . .",
"ncDNA amounts to more than 98% of the human DNA genome . . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression.",
"ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information.",
"DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 .",
"Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 . The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat . . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?",
"What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners? According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it \"forgotten\" to be removed? Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011 . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA.",
"Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus Figure 1 ? Patel et al. . demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 .",
"demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions Simon and Zimmerly, 2008; Lescot et al., 2016 . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world.",
"It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H RNase H is an essential enzyme of retroviruses . . The RNase H turned out to be one of the five most frequent and ancient proteins Ma et al., 2008 that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions . . Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 .",
". Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus TMV , and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 .",
"The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 . They look like \"frozen\" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element. Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 .",
"Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as \"living fossils\" Diener, 2016 Figure 2 . From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides.",
"From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as \"sponges\" for other regulatory RNAs. information in the form of hairpin-loops . .",
"information in the form of hairpin-loops . . They can generate copies of themselves in the appropriate environment. They were designated as the \"frontiers of life\" . . The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 Pennazio and Roggero, 2000 . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens.",
"Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid ASBVd . Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle. Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss.",
"Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle.",
"Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA CarSV RNA cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners?",
"This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation. The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs.",
"The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules . . Their structure is reminiscent of catalytically active ribozymes. There is an exceptional viroid that gained coding information and entered the human liver Taylor, 2009 . The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides.",
"The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly.",
"Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus HBV , which delivers the coat Taylor, 2009 Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist? In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct.",
"In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as \"error catastrophe, \" which prevents survival Eigen, 1971 Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions.",
"The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma Crick, 1968 . Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions.",
"Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world.",
"Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed \"virus-first, \" such as \"protein-first\", \"metabolism-fist\" or the \"lipid world\" Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012 . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing . . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA.",
". Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert Crick, 1968; Gilbert, 1986 . What a vision! Our world was almost 50 years later defined as \"RNAprotein\" world Altman, 2013 . One can speculate our world was built of ribozymes or viroids, which means \"viruses first.\" ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today.",
"ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase.",
"Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 !",
"This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 .",
"The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity. The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.",
"The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again. These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out .",
"This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out . . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV Taylor, 2009 . An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth.",
"Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals core sequences as a stable composition Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 .",
"Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment. The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . .",
"Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 .",
"The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 . To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.",
"Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions. The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes.",
"Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism.",
"They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information Nasir and Caetano-Anolles, 2015 . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer HGT La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018 . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes.",
"A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million mio basepairs Mitch, 2017 . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia . . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp .",
". The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp . Figure 3 . The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses.",
"They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive La Scola et al., 2008; Fischer and Hackl, 2016 . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 .",
"The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 . Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter . . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today.",
". The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria see below . Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990.",
"Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful?",
"It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants Gibson et al., 2008 Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans.",
"His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt . . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name . . The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA.",
". The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length Figure 3 . The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses!",
"There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence .",
"Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence . . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses ERVs and related retroelements REs . . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome.",
"In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV HERV elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats LTR that act as promoters . . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence Temin, 1985 . The actual number is about 45% as estimated today . .",
"The actual number is about 45% as estimated today . . In some genes such as the Protein Kinase Inhibitor B PKIB gene we determined about 70% retrovirusrelated sequences Moelling and Broecker, 2015 . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago MYA Hayward, 2017 . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp.",
"Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 .",
"It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 . A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses.",
"They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses? These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 .",
"REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017 . This gene duplication dates back to 500 mio years Cotton and Page, 2005 . Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 .",
"Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred. Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer .",
"Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer . . HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name.",
"The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as \"necessary factor\" Flint, 2015 . Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally.",
"Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections . . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose.",
"This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described Galvani and Slatkin, 2003 . Viruses have been proven to be drivers of evolution Villarreal and Witzany, 2010 , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . .",
"In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity Villarreal, 2009 . Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 .",
"Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity.",
"The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus GALV . Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations.",
"Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus JSRV , whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by \"superinfection exclusion\" Tarlinton, 2012 . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . .",
"The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . . Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors Villarreal, 2011 . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity Charpentier and Doudna, 2013 . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle.",
"It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as \"superinfection exclusion\" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections.",
"This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text. to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 .",
"to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of \"endogenization\" of incoming genetic material for subsequent exclusion. One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations?",
"There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations? The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection.",
"The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by \"downstream promotion\" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer . . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless.",
"As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon Broecker et al., 2016b . As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency .",
"As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency . . Thus, the role of ERVs may be more complex than we presently know. Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are \"locked\" inside the cells and are major drivers of the increase of genetic complexity Cordaux and Batzer, 2009 . One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . .",
"One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease Beltz, 2018 . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection Katzourakis and Gifford, 2010 . There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 .",
"This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 . An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago BYA Embley and Martin, 2006 . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet.",
"Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii the causative agent of Q fever , Mycobacterium leprae, M. tuberculosis, and M. mycoides . . The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes.",
"Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life.",
"Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution . . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts. Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells?",
"Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic? Viruses are minimalistic in their composition and must have undergone stringent gene reductions Flint, 2015 . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information.",
"Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . .",
"Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . . These oncogenes were in most cases taken up by the retroviruses at the expense of structural gag , replicating pol or envelope env genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells.",
"They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src for sarcoma oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome Broecker et al., 2016a . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes. There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis .",
"There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis . . Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants.",
"In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species. Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution.",
"Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 .",
"Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex.",
"By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication Moelling, 2012 Moelling, , 2013 . Therefore, the question may be repeated: \"Are viruses our oldest ancestors?\" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong.",
"Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled \"Viruses: More Friends Than Foes\" Moelling, 2017 . The scenario described here focuses on viruses as drivers of evolution. The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . .",
"Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life. The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence."
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What contributed to a large part of mammalian genomic sequence? | Retroviruses | [
"The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution?",
"Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes.",
"Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life.",
"Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.” Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes . . Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids.",
". Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules aptamers , 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds.",
"The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube Lincoln and Joyce, 2009 . Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity.",
"Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid PNA , with more stable peptide bonds instead of phosphodiester bonds Zhang and Cech, 1997; Joyce, 2002 . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides Wilson and Szostak, 1999 . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes.",
"Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs Orgel, 2004 . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids.",
"They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis Meierhenrich, 2008 . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started . . There, temperature gradients and clay containing minerals such as magnesium or manganese are available.",
". There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication . . tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code.",
". tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: \"The ribosome is a ribozyme\" Cech, 2000 , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 .",
"Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 . Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro . . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV . . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . .",
". Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases . . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes Harish and Caetano-Anolles, 2012 . These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase.",
"Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme a reductase as today, and allow for DNA polymerization Wilson and Szostak, 1999; Joyce, 2002 . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin Moelling, 2012 Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . .",
"tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins Moelling and Broecker, 2015; Moelling et al., 2017 Figure 1 . It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the \"Encyclopedia of DNA Elements\" ENCODE project. ncDNA amounts to more than 98% of the human DNA genome . .",
"ncDNA amounts to more than 98% of the human DNA genome . . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression.",
"ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information.",
"DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 .",
"Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 . The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat . . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?",
"What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners? According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it \"forgotten\" to be removed? Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011 . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA.",
"Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus Figure 1 ? Patel et al. . demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 .",
"demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions Simon and Zimmerly, 2008; Lescot et al., 2016 . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world.",
"It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H RNase H is an essential enzyme of retroviruses . . The RNase H turned out to be one of the five most frequent and ancient proteins Ma et al., 2008 that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions . . Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 .",
". Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus TMV , and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 .",
"The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 . They look like \"frozen\" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element. Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 .",
"Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as \"living fossils\" Diener, 2016 Figure 2 . From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides.",
"From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as \"sponges\" for other regulatory RNAs. information in the form of hairpin-loops . .",
"information in the form of hairpin-loops . . They can generate copies of themselves in the appropriate environment. They were designated as the \"frontiers of life\" . . The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 Pennazio and Roggero, 2000 . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens.",
"Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid ASBVd . Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle. Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss.",
"Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle.",
"Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA CarSV RNA cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners?",
"This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation. The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs.",
"The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules . . Their structure is reminiscent of catalytically active ribozymes. There is an exceptional viroid that gained coding information and entered the human liver Taylor, 2009 . The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides.",
"The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly.",
"Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus HBV , which delivers the coat Taylor, 2009 Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist? In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct.",
"In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as \"error catastrophe, \" which prevents survival Eigen, 1971 Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions.",
"The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma Crick, 1968 . Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions.",
"Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world.",
"Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed \"virus-first, \" such as \"protein-first\", \"metabolism-fist\" or the \"lipid world\" Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012 . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing . . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA.",
". Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert Crick, 1968; Gilbert, 1986 . What a vision! Our world was almost 50 years later defined as \"RNAprotein\" world Altman, 2013 . One can speculate our world was built of ribozymes or viroids, which means \"viruses first.\" ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today.",
"ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase.",
"Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 !",
"This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 .",
"The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity. The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.",
"The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again. These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out .",
"This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out . . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV Taylor, 2009 . An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth.",
"Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals core sequences as a stable composition Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 .",
"Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment. The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . .",
"Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 .",
"The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 . To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.",
"Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions. The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes.",
"Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism.",
"They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information Nasir and Caetano-Anolles, 2015 . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer HGT La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018 . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes.",
"A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million mio basepairs Mitch, 2017 . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia . . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp .",
". The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp . Figure 3 . The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses.",
"They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive La Scola et al., 2008; Fischer and Hackl, 2016 . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 .",
"The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 . Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter . . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today.",
". The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria see below . Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990.",
"Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful?",
"It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants Gibson et al., 2008 Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans.",
"His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt . . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name . . The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA.",
". The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length Figure 3 . The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses!",
"There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence .",
"Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence . . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses ERVs and related retroelements REs . . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome.",
"In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV HERV elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats LTR that act as promoters . . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence Temin, 1985 . The actual number is about 45% as estimated today . .",
"The actual number is about 45% as estimated today . . In some genes such as the Protein Kinase Inhibitor B PKIB gene we determined about 70% retrovirusrelated sequences Moelling and Broecker, 2015 . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago MYA Hayward, 2017 . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp.",
"Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 .",
"It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 . A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses.",
"They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses? These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 .",
"REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017 . This gene duplication dates back to 500 mio years Cotton and Page, 2005 . Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 .",
"Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred. Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer .",
"Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer . . HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name.",
"The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as \"necessary factor\" Flint, 2015 . Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally.",
"Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections . . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose.",
"This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described Galvani and Slatkin, 2003 . Viruses have been proven to be drivers of evolution Villarreal and Witzany, 2010 , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . .",
"In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity Villarreal, 2009 . Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 .",
"Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity.",
"The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus GALV . Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations.",
"Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus JSRV , whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by \"superinfection exclusion\" Tarlinton, 2012 . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . .",
"The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . . Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors Villarreal, 2011 . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity Charpentier and Doudna, 2013 . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle.",
"It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as \"superinfection exclusion\" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections.",
"This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text. to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 .",
"to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of \"endogenization\" of incoming genetic material for subsequent exclusion. One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations?",
"There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations? The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection.",
"The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by \"downstream promotion\" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer . . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless.",
"As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon Broecker et al., 2016b . As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency .",
"As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency . . Thus, the role of ERVs may be more complex than we presently know. Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are \"locked\" inside the cells and are major drivers of the increase of genetic complexity Cordaux and Batzer, 2009 . One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . .",
"One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease Beltz, 2018 . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection Katzourakis and Gifford, 2010 . There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 .",
"This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 . An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago BYA Embley and Martin, 2006 . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet.",
"Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii the causative agent of Q fever , Mycobacterium leprae, M. tuberculosis, and M. mycoides . . The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes.",
"Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life.",
"Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution . . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts. Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells?",
"Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic? Viruses are minimalistic in their composition and must have undergone stringent gene reductions Flint, 2015 . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information.",
"Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . .",
"Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . . These oncogenes were in most cases taken up by the retroviruses at the expense of structural gag , replicating pol or envelope env genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells.",
"They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src for sarcoma oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome Broecker et al., 2016a . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes. There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis .",
"There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis . . Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants.",
"In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species. Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution.",
"Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 .",
"Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex.",
"By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication Moelling, 2012 Moelling, , 2013 . Therefore, the question may be repeated: \"Are viruses our oldest ancestors?\" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong.",
"Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled \"Viruses: More Friends Than Foes\" Moelling, 2017 . The scenario described here focuses on viruses as drivers of evolution. The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . .",
"Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life. The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence."
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What were the earliest replicating entities that fulfill several criteria for life? | ribozymes or viroids | [
"The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution?",
"Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes.",
"Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life.",
"Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.” Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes . . Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids.",
". Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules aptamers , 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds.",
"The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube Lincoln and Joyce, 2009 . Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity.",
"Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid PNA , with more stable peptide bonds instead of phosphodiester bonds Zhang and Cech, 1997; Joyce, 2002 . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides Wilson and Szostak, 1999 . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes.",
"Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs Orgel, 2004 . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids.",
"They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis Meierhenrich, 2008 . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started . . There, temperature gradients and clay containing minerals such as magnesium or manganese are available.",
". There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication . . tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code.",
". tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: \"The ribosome is a ribozyme\" Cech, 2000 , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 .",
"Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 . Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro . . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV . . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . .",
". Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases . . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes Harish and Caetano-Anolles, 2012 . These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase.",
"Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme a reductase as today, and allow for DNA polymerization Wilson and Szostak, 1999; Joyce, 2002 . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin Moelling, 2012 Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . .",
"tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins Moelling and Broecker, 2015; Moelling et al., 2017 Figure 1 . It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the \"Encyclopedia of DNA Elements\" ENCODE project. ncDNA amounts to more than 98% of the human DNA genome . .",
"ncDNA amounts to more than 98% of the human DNA genome . . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression.",
"ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information.",
"DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 .",
"Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 . The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat . . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?",
"What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners? According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it \"forgotten\" to be removed? Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011 . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA.",
"Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus Figure 1 ? Patel et al. . demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 .",
"demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions Simon and Zimmerly, 2008; Lescot et al., 2016 . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world.",
"It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H RNase H is an essential enzyme of retroviruses . . The RNase H turned out to be one of the five most frequent and ancient proteins Ma et al., 2008 that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions . . Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 .",
". Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus TMV , and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 .",
"The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 . They look like \"frozen\" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element. Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 .",
"Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as \"living fossils\" Diener, 2016 Figure 2 . From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides.",
"From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as \"sponges\" for other regulatory RNAs. information in the form of hairpin-loops . .",
"information in the form of hairpin-loops . . They can generate copies of themselves in the appropriate environment. They were designated as the \"frontiers of life\" . . The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 Pennazio and Roggero, 2000 . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens.",
"Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid ASBVd . Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle. Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss.",
"Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle.",
"Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA CarSV RNA cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners?",
"This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation. The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs.",
"The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules . . Their structure is reminiscent of catalytically active ribozymes. There is an exceptional viroid that gained coding information and entered the human liver Taylor, 2009 . The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides.",
"The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly.",
"Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus HBV , which delivers the coat Taylor, 2009 Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist? In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct.",
"In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as \"error catastrophe, \" which prevents survival Eigen, 1971 Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions.",
"The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma Crick, 1968 . Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions.",
"Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world.",
"Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed \"virus-first, \" such as \"protein-first\", \"metabolism-fist\" or the \"lipid world\" Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012 . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing . . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA.",
". Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert Crick, 1968; Gilbert, 1986 . What a vision! Our world was almost 50 years later defined as \"RNAprotein\" world Altman, 2013 . One can speculate our world was built of ribozymes or viroids, which means \"viruses first.\" ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today.",
"ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase.",
"Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 !",
"This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 .",
"The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity. The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.",
"The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again. These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out .",
"This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out . . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV Taylor, 2009 . An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth.",
"Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals core sequences as a stable composition Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 .",
"Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment. The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . .",
"Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 .",
"The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 . To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.",
"Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions. The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes.",
"Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism.",
"They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information Nasir and Caetano-Anolles, 2015 . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer HGT La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018 . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes.",
"A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million mio basepairs Mitch, 2017 . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia . . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp .",
". The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp . Figure 3 . The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses.",
"They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive La Scola et al., 2008; Fischer and Hackl, 2016 . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 .",
"The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 . Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter . . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today.",
". The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria see below . Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990.",
"Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful?",
"It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants Gibson et al., 2008 Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans.",
"His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt . . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name . . The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA.",
". The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length Figure 3 . The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses!",
"There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence .",
"Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence . . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses ERVs and related retroelements REs . . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome.",
"In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV HERV elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats LTR that act as promoters . . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence Temin, 1985 . The actual number is about 45% as estimated today . .",
"The actual number is about 45% as estimated today . . In some genes such as the Protein Kinase Inhibitor B PKIB gene we determined about 70% retrovirusrelated sequences Moelling and Broecker, 2015 . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago MYA Hayward, 2017 . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp.",
"Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 .",
"It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 . A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses.",
"They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses? These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 .",
"REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017 . This gene duplication dates back to 500 mio years Cotton and Page, 2005 . Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 .",
"Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred. Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer .",
"Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer . . HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name.",
"The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as \"necessary factor\" Flint, 2015 . Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally.",
"Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections . . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose.",
"This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described Galvani and Slatkin, 2003 . Viruses have been proven to be drivers of evolution Villarreal and Witzany, 2010 , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . .",
"In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity Villarreal, 2009 . Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 .",
"Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity.",
"The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus GALV . Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations.",
"Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus JSRV , whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by \"superinfection exclusion\" Tarlinton, 2012 . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . .",
"The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . . Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors Villarreal, 2011 . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity Charpentier and Doudna, 2013 . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle.",
"It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as \"superinfection exclusion\" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections.",
"This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text. to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 .",
"to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of \"endogenization\" of incoming genetic material for subsequent exclusion. One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations?",
"There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations? The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection.",
"The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by \"downstream promotion\" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer . . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless.",
"As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon Broecker et al., 2016b . As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency .",
"As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency . . Thus, the role of ERVs may be more complex than we presently know. Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are \"locked\" inside the cells and are major drivers of the increase of genetic complexity Cordaux and Batzer, 2009 . One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . .",
"One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease Beltz, 2018 . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection Katzourakis and Gifford, 2010 . There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 .",
"This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 . An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago BYA Embley and Martin, 2006 . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet.",
"Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii the causative agent of Q fever , Mycobacterium leprae, M. tuberculosis, and M. mycoides . . The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes.",
"Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life.",
"Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution . . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts. Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells?",
"Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic? Viruses are minimalistic in their composition and must have undergone stringent gene reductions Flint, 2015 . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information.",
"Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . .",
"Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . . These oncogenes were in most cases taken up by the retroviruses at the expense of structural gag , replicating pol or envelope env genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells.",
"They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src for sarcoma oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome Broecker et al., 2016a . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes. There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis .",
"There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis . . Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants.",
"In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species. Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution.",
"Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 .",
"Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex.",
"By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication Moelling, 2012 Moelling, , 2013 . Therefore, the question may be repeated: \"Are viruses our oldest ancestors?\" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong.",
"Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled \"Viruses: More Friends Than Foes\" Moelling, 2017 . The scenario described here focuses on viruses as drivers of evolution. The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . .",
"Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life. The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence."
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What are some examples of autonomous bacteria that lost their independence and became intracellular parasites or endosymbionts? | mitochondria, chloroplasts, Rickettsia | [
"The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution?",
"Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes.",
"Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life.",
"Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.” Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes . . Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids.",
". Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules aptamers , 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds.",
"The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube Lincoln and Joyce, 2009 . Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity.",
"Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid PNA , with more stable peptide bonds instead of phosphodiester bonds Zhang and Cech, 1997; Joyce, 2002 . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides Wilson and Szostak, 1999 . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes.",
"Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs Orgel, 2004 . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids.",
"They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis Meierhenrich, 2008 . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started . . There, temperature gradients and clay containing minerals such as magnesium or manganese are available.",
". There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication . . tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code.",
". tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: \"The ribosome is a ribozyme\" Cech, 2000 , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 .",
"Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 . Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro . . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV . . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . .",
". Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases . . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes Harish and Caetano-Anolles, 2012 . These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase.",
"Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme a reductase as today, and allow for DNA polymerization Wilson and Szostak, 1999; Joyce, 2002 . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin Moelling, 2012 Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . .",
"tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins Moelling and Broecker, 2015; Moelling et al., 2017 Figure 1 . It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the \"Encyclopedia of DNA Elements\" ENCODE project. ncDNA amounts to more than 98% of the human DNA genome . .",
"ncDNA amounts to more than 98% of the human DNA genome . . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression.",
"ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information.",
"DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 .",
"Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 . The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat . . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?",
"What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners? According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it \"forgotten\" to be removed? Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011 . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA.",
"Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus Figure 1 ? Patel et al. . demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 .",
"demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions Simon and Zimmerly, 2008; Lescot et al., 2016 . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world.",
"It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H RNase H is an essential enzyme of retroviruses . . The RNase H turned out to be one of the five most frequent and ancient proteins Ma et al., 2008 that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions . . Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 .",
". Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus TMV , and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 .",
"The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 . They look like \"frozen\" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element. Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 .",
"Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as \"living fossils\" Diener, 2016 Figure 2 . From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides.",
"From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as \"sponges\" for other regulatory RNAs. information in the form of hairpin-loops . .",
"information in the form of hairpin-loops . . They can generate copies of themselves in the appropriate environment. They were designated as the \"frontiers of life\" . . The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 Pennazio and Roggero, 2000 . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens.",
"Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid ASBVd . Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle. Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss.",
"Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle.",
"Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA CarSV RNA cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners?",
"This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation. The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs.",
"The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules . . Their structure is reminiscent of catalytically active ribozymes. There is an exceptional viroid that gained coding information and entered the human liver Taylor, 2009 . The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides.",
"The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly.",
"Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus HBV , which delivers the coat Taylor, 2009 Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist? In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct.",
"In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as \"error catastrophe, \" which prevents survival Eigen, 1971 Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions.",
"The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma Crick, 1968 . Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions.",
"Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world.",
"Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed \"virus-first, \" such as \"protein-first\", \"metabolism-fist\" or the \"lipid world\" Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012 . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing . . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA.",
". Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert Crick, 1968; Gilbert, 1986 . What a vision! Our world was almost 50 years later defined as \"RNAprotein\" world Altman, 2013 . One can speculate our world was built of ribozymes or viroids, which means \"viruses first.\" ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today.",
"ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase.",
"Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 !",
"This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 .",
"The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity. The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.",
"The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again. These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out .",
"This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out . . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV Taylor, 2009 . An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth.",
"Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals core sequences as a stable composition Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 .",
"Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment. The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . .",
"Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 .",
"The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 . To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.",
"Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions. The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes.",
"Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism.",
"They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information Nasir and Caetano-Anolles, 2015 . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer HGT La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018 . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes.",
"A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million mio basepairs Mitch, 2017 . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia . . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp .",
". The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp . Figure 3 . The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses.",
"They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive La Scola et al., 2008; Fischer and Hackl, 2016 . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 .",
"The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 . Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter . . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today.",
". The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria see below . Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990.",
"Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful?",
"It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants Gibson et al., 2008 Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans.",
"His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt . . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name . . The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA.",
". The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length Figure 3 . The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses!",
"There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence .",
"Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence . . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses ERVs and related retroelements REs . . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome.",
"In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV HERV elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats LTR that act as promoters . . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence Temin, 1985 . The actual number is about 45% as estimated today . .",
"The actual number is about 45% as estimated today . . In some genes such as the Protein Kinase Inhibitor B PKIB gene we determined about 70% retrovirusrelated sequences Moelling and Broecker, 2015 . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago MYA Hayward, 2017 . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp.",
"Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 .",
"It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 . A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses.",
"They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses? These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 .",
"REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017 . This gene duplication dates back to 500 mio years Cotton and Page, 2005 . Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 .",
"Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred. Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer .",
"Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer . . HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name.",
"The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as \"necessary factor\" Flint, 2015 . Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally.",
"Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections . . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose.",
"This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described Galvani and Slatkin, 2003 . Viruses have been proven to be drivers of evolution Villarreal and Witzany, 2010 , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . .",
"In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity Villarreal, 2009 . Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 .",
"Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity.",
"The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus GALV . Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations.",
"Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus JSRV , whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by \"superinfection exclusion\" Tarlinton, 2012 . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . .",
"The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . . Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors Villarreal, 2011 . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity Charpentier and Doudna, 2013 . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle.",
"It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as \"superinfection exclusion\" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections.",
"This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text. to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 .",
"to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of \"endogenization\" of incoming genetic material for subsequent exclusion. One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations?",
"There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations? The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection.",
"The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by \"downstream promotion\" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer . . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless.",
"As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon Broecker et al., 2016b . As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency .",
"As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency . . Thus, the role of ERVs may be more complex than we presently know. Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are \"locked\" inside the cells and are major drivers of the increase of genetic complexity Cordaux and Batzer, 2009 . One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . .",
"One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease Beltz, 2018 . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection Katzourakis and Gifford, 2010 . There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 .",
"This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 . An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago BYA Embley and Martin, 2006 . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet.",
"Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii the causative agent of Q fever , Mycobacterium leprae, M. tuberculosis, and M. mycoides . . The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes.",
"Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life.",
"Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution . . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts. Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells?",
"Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic? Viruses are minimalistic in their composition and must have undergone stringent gene reductions Flint, 2015 . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information.",
"Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . .",
"Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . . These oncogenes were in most cases taken up by the retroviruses at the expense of structural gag , replicating pol or envelope env genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells.",
"They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src for sarcoma oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome Broecker et al., 2016a . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes. There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis .",
"There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis . . Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants.",
"In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species. Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution.",
"Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 .",
"Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex.",
"By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication Moelling, 2012 Moelling, , 2013 . Therefore, the question may be repeated: \"Are viruses our oldest ancestors?\" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong.",
"Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled \"Viruses: More Friends Than Foes\" Moelling, 2017 . The scenario described here focuses on viruses as drivers of evolution. The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . .",
"Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life. The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence."
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What entities with no genes satisfy the criteria for life? | ribozymes, catalytic RNAs closely related to viroids | [
"The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution?",
"Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes.",
"Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life.",
"Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.” Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes . . Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids.",
". Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules aptamers , 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds.",
"The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube Lincoln and Joyce, 2009 . Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity.",
"Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid PNA , with more stable peptide bonds instead of phosphodiester bonds Zhang and Cech, 1997; Joyce, 2002 . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides Wilson and Szostak, 1999 . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes.",
"Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs Orgel, 2004 . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids.",
"They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis Meierhenrich, 2008 . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started . . There, temperature gradients and clay containing minerals such as magnesium or manganese are available.",
". There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication . . tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code.",
". tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: \"The ribosome is a ribozyme\" Cech, 2000 , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 .",
"Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 . Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro . . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV . . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . .",
". Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases . . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes Harish and Caetano-Anolles, 2012 . These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase.",
"Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme a reductase as today, and allow for DNA polymerization Wilson and Szostak, 1999; Joyce, 2002 . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin Moelling, 2012 Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . .",
"tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins Moelling and Broecker, 2015; Moelling et al., 2017 Figure 1 . It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the \"Encyclopedia of DNA Elements\" ENCODE project. ncDNA amounts to more than 98% of the human DNA genome . .",
"ncDNA amounts to more than 98% of the human DNA genome . . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression.",
"ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information.",
"DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 .",
"Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 . The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat . . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?",
"What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners? According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it \"forgotten\" to be removed? Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011 . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA.",
"Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus Figure 1 ? Patel et al. . demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 .",
"demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions Simon and Zimmerly, 2008; Lescot et al., 2016 . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world.",
"It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H RNase H is an essential enzyme of retroviruses . . The RNase H turned out to be one of the five most frequent and ancient proteins Ma et al., 2008 that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions . . Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 .",
". Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus TMV , and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 .",
"The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 . They look like \"frozen\" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element. Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 .",
"Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as \"living fossils\" Diener, 2016 Figure 2 . From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides.",
"From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as \"sponges\" for other regulatory RNAs. information in the form of hairpin-loops . .",
"information in the form of hairpin-loops . . They can generate copies of themselves in the appropriate environment. They were designated as the \"frontiers of life\" . . The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 Pennazio and Roggero, 2000 . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens.",
"Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid ASBVd . Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle. Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss.",
"Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle.",
"Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA CarSV RNA cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners?",
"This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation. The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs.",
"The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules . . Their structure is reminiscent of catalytically active ribozymes. There is an exceptional viroid that gained coding information and entered the human liver Taylor, 2009 . The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides.",
"The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly.",
"Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus HBV , which delivers the coat Taylor, 2009 Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist? In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct.",
"In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as \"error catastrophe, \" which prevents survival Eigen, 1971 Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions.",
"The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma Crick, 1968 . Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions.",
"Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world.",
"Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed \"virus-first, \" such as \"protein-first\", \"metabolism-fist\" or the \"lipid world\" Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012 . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing . . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA.",
". Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert Crick, 1968; Gilbert, 1986 . What a vision! Our world was almost 50 years later defined as \"RNAprotein\" world Altman, 2013 . One can speculate our world was built of ribozymes or viroids, which means \"viruses first.\" ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today.",
"ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase.",
"Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 !",
"This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 .",
"The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity. The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.",
"The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again. These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out .",
"This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out . . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV Taylor, 2009 . An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth.",
"Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals core sequences as a stable composition Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 .",
"Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment. The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . .",
"Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 .",
"The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 . To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.",
"Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions. The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes.",
"Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism.",
"They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information Nasir and Caetano-Anolles, 2015 . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer HGT La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018 . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes.",
"A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million mio basepairs Mitch, 2017 . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia . . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp .",
". The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp . Figure 3 . The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses.",
"They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive La Scola et al., 2008; Fischer and Hackl, 2016 . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 .",
"The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 . Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter . . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today.",
". The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria see below . Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990.",
"Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful?",
"It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants Gibson et al., 2008 Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans.",
"His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt . . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name . . The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA.",
". The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length Figure 3 . The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses!",
"There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence .",
"Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence . . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses ERVs and related retroelements REs . . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome.",
"In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV HERV elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats LTR that act as promoters . . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence Temin, 1985 . The actual number is about 45% as estimated today . .",
"The actual number is about 45% as estimated today . . In some genes such as the Protein Kinase Inhibitor B PKIB gene we determined about 70% retrovirusrelated sequences Moelling and Broecker, 2015 . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago MYA Hayward, 2017 . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp.",
"Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 .",
"It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 . A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses.",
"They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses? These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 .",
"REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017 . This gene duplication dates back to 500 mio years Cotton and Page, 2005 . Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 .",
"Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred. Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer .",
"Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer . . HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name.",
"The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as \"necessary factor\" Flint, 2015 . Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally.",
"Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections . . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose.",
"This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described Galvani and Slatkin, 2003 . Viruses have been proven to be drivers of evolution Villarreal and Witzany, 2010 , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . .",
"In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity Villarreal, 2009 . Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 .",
"Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity.",
"The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus GALV . Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations.",
"Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus JSRV , whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by \"superinfection exclusion\" Tarlinton, 2012 . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . .",
"The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . . Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors Villarreal, 2011 . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity Charpentier and Doudna, 2013 . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle.",
"It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as \"superinfection exclusion\" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections.",
"This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text. to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 .",
"to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of \"endogenization\" of incoming genetic material for subsequent exclusion. One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations?",
"There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations? The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection.",
"The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by \"downstream promotion\" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer . . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless.",
"As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon Broecker et al., 2016b . As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency .",
"As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency . . Thus, the role of ERVs may be more complex than we presently know. Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are \"locked\" inside the cells and are major drivers of the increase of genetic complexity Cordaux and Batzer, 2009 . One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . .",
"One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease Beltz, 2018 . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection Katzourakis and Gifford, 2010 . There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 .",
"This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 . An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago BYA Embley and Martin, 2006 . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet.",
"Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii the causative agent of Q fever , Mycobacterium leprae, M. tuberculosis, and M. mycoides . . The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes.",
"Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life.",
"Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution . . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts. Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells?",
"Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic? Viruses are minimalistic in their composition and must have undergone stringent gene reductions Flint, 2015 . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information.",
"Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . .",
"Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . . These oncogenes were in most cases taken up by the retroviruses at the expense of structural gag , replicating pol or envelope env genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells.",
"They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src for sarcoma oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome Broecker et al., 2016a . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes. There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis .",
"There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis . . Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants.",
"In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species. Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution.",
"Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 .",
"Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex.",
"By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication Moelling, 2012 Moelling, , 2013 . Therefore, the question may be repeated: \"Are viruses our oldest ancestors?\" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong.",
"Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled \"Viruses: More Friends Than Foes\" Moelling, 2017 . The scenario described here focuses on viruses as drivers of evolution. The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . .",
"Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life. The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence."
] | 1,690 | 1,179 |
Which group of RNA quasispecies satisfy criteria for life? | catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 ( | [
"The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution?",
"Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes.",
"Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life.",
"Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.” Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes . . Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids.",
". Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules aptamers , 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds.",
"The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube Lincoln and Joyce, 2009 . Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity.",
"Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid PNA , with more stable peptide bonds instead of phosphodiester bonds Zhang and Cech, 1997; Joyce, 2002 . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides Wilson and Szostak, 1999 . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes.",
"Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs Orgel, 2004 . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids.",
"They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis Meierhenrich, 2008 . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started . . There, temperature gradients and clay containing minerals such as magnesium or manganese are available.",
". There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication . . tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code.",
". tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: \"The ribosome is a ribozyme\" Cech, 2000 , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 .",
"Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 . Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro . . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV . . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . .",
". Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases . . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes Harish and Caetano-Anolles, 2012 . These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase.",
"Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme a reductase as today, and allow for DNA polymerization Wilson and Szostak, 1999; Joyce, 2002 . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin Moelling, 2012 Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . .",
"tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins Moelling and Broecker, 2015; Moelling et al., 2017 Figure 1 . It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the \"Encyclopedia of DNA Elements\" ENCODE project. ncDNA amounts to more than 98% of the human DNA genome . .",
"ncDNA amounts to more than 98% of the human DNA genome . . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression.",
"ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information.",
"DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 .",
"Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 . The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat . . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?",
"What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners? According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it \"forgotten\" to be removed? Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011 . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA.",
"Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus Figure 1 ? Patel et al. . demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 .",
"demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions Simon and Zimmerly, 2008; Lescot et al., 2016 . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world.",
"It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H RNase H is an essential enzyme of retroviruses . . The RNase H turned out to be one of the five most frequent and ancient proteins Ma et al., 2008 that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions . . Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 .",
". Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus TMV , and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 .",
"The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 . They look like \"frozen\" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element. Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 .",
"Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as \"living fossils\" Diener, 2016 Figure 2 . From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides.",
"From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as \"sponges\" for other regulatory RNAs. information in the form of hairpin-loops . .",
"information in the form of hairpin-loops . . They can generate copies of themselves in the appropriate environment. They were designated as the \"frontiers of life\" . . The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 Pennazio and Roggero, 2000 . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens.",
"Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid ASBVd . Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle. Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss.",
"Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle.",
"Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA CarSV RNA cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners?",
"This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation. The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs.",
"The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules . . Their structure is reminiscent of catalytically active ribozymes. There is an exceptional viroid that gained coding information and entered the human liver Taylor, 2009 . The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides.",
"The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly.",
"Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus HBV , which delivers the coat Taylor, 2009 Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist? In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct.",
"In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as \"error catastrophe, \" which prevents survival Eigen, 1971 Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions.",
"The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma Crick, 1968 . Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions.",
"Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world.",
"Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed \"virus-first, \" such as \"protein-first\", \"metabolism-fist\" or the \"lipid world\" Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012 . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing . . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA.",
". Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert Crick, 1968; Gilbert, 1986 . What a vision! Our world was almost 50 years later defined as \"RNAprotein\" world Altman, 2013 . One can speculate our world was built of ribozymes or viroids, which means \"viruses first.\" ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today.",
"ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase.",
"Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 !",
"This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 .",
"The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity. The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.",
"The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again. These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out .",
"This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out . . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV Taylor, 2009 . An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth.",
"Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals core sequences as a stable composition Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 .",
"Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment. The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . .",
"Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 .",
"The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 . To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.",
"Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions. The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes.",
"Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism.",
"They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information Nasir and Caetano-Anolles, 2015 . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer HGT La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018 . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes.",
"A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million mio basepairs Mitch, 2017 . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia . . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp .",
". The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp . Figure 3 . The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses.",
"They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive La Scola et al., 2008; Fischer and Hackl, 2016 . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 .",
"The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 . Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter . . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today.",
". The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria see below . Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990.",
"Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful?",
"It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants Gibson et al., 2008 Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans.",
"His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt . . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name . . The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA.",
". The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length Figure 3 . The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses!",
"There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence .",
"Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence . . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses ERVs and related retroelements REs . . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome.",
"In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV HERV elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats LTR that act as promoters . . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence Temin, 1985 . The actual number is about 45% as estimated today . .",
"The actual number is about 45% as estimated today . . In some genes such as the Protein Kinase Inhibitor B PKIB gene we determined about 70% retrovirusrelated sequences Moelling and Broecker, 2015 . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago MYA Hayward, 2017 . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp.",
"Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 .",
"It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 . A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses.",
"They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses? These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 .",
"REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017 . This gene duplication dates back to 500 mio years Cotton and Page, 2005 . Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 .",
"Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred. Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer .",
"Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer . . HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name.",
"The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as \"necessary factor\" Flint, 2015 . Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally.",
"Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections . . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose.",
"This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described Galvani and Slatkin, 2003 . Viruses have been proven to be drivers of evolution Villarreal and Witzany, 2010 , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . .",
"In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity Villarreal, 2009 . Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 .",
"Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity.",
"The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus GALV . Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations.",
"Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus JSRV , whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by \"superinfection exclusion\" Tarlinton, 2012 . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . .",
"The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . . Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors Villarreal, 2011 . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity Charpentier and Doudna, 2013 . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle.",
"It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as \"superinfection exclusion\" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections.",
"This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text. to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 .",
"to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of \"endogenization\" of incoming genetic material for subsequent exclusion. One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations?",
"There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations? The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection.",
"The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by \"downstream promotion\" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer . . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless.",
"As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon Broecker et al., 2016b . As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency .",
"As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency . . Thus, the role of ERVs may be more complex than we presently know. Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are \"locked\" inside the cells and are major drivers of the increase of genetic complexity Cordaux and Batzer, 2009 . One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . .",
"One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease Beltz, 2018 . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection Katzourakis and Gifford, 2010 . There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 .",
"This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 . An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago BYA Embley and Martin, 2006 . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet.",
"Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii the causative agent of Q fever , Mycobacterium leprae, M. tuberculosis, and M. mycoides . . The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes.",
"Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life.",
"Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution . . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts. Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells?",
"Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic? Viruses are minimalistic in their composition and must have undergone stringent gene reductions Flint, 2015 . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information.",
"Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . .",
"Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . . These oncogenes were in most cases taken up by the retroviruses at the expense of structural gag , replicating pol or envelope env genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells.",
"They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src for sarcoma oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome Broecker et al., 2016a . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes. There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis .",
"There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis . . Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants.",
"In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species. Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution.",
"Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 .",
"Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex.",
"By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication Moelling, 2012 Moelling, , 2013 . Therefore, the question may be repeated: \"Are viruses our oldest ancestors?\" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong.",
"Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled \"Viruses: More Friends Than Foes\" Moelling, 2017 . The scenario described here focuses on viruses as drivers of evolution. The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . .",
"Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life. The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence."
] | 1,690 | 1,180 |
How are the ribozymes able to replicate, join and create peptide bonds? | They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. | [
"The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution?",
"Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes.",
"Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life.",
"Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.” Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes . . Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids.",
". Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules aptamers , 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds.",
"The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube Lincoln and Joyce, 2009 . Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity.",
"Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid PNA , with more stable peptide bonds instead of phosphodiester bonds Zhang and Cech, 1997; Joyce, 2002 . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides Wilson and Szostak, 1999 . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes.",
"Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs Orgel, 2004 . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids.",
"They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis Meierhenrich, 2008 . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started . . There, temperature gradients and clay containing minerals such as magnesium or manganese are available.",
". There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication . . tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code.",
". tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: \"The ribosome is a ribozyme\" Cech, 2000 , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 .",
"Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 . Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro . . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV . . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . .",
". Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases . . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes Harish and Caetano-Anolles, 2012 . These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase.",
"Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme a reductase as today, and allow for DNA polymerization Wilson and Szostak, 1999; Joyce, 2002 . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin Moelling, 2012 Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . .",
"tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins Moelling and Broecker, 2015; Moelling et al., 2017 Figure 1 . It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the \"Encyclopedia of DNA Elements\" ENCODE project. ncDNA amounts to more than 98% of the human DNA genome . .",
"ncDNA amounts to more than 98% of the human DNA genome . . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression.",
"ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information.",
"DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 .",
"Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 . The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat . . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?",
"What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners? According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it \"forgotten\" to be removed? Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011 . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA.",
"Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus Figure 1 ? Patel et al. . demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 .",
"demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions Simon and Zimmerly, 2008; Lescot et al., 2016 . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world.",
"It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H RNase H is an essential enzyme of retroviruses . . The RNase H turned out to be one of the five most frequent and ancient proteins Ma et al., 2008 that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions . . Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 .",
". Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus TMV , and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 .",
"The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 . They look like \"frozen\" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element. Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 .",
"Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as \"living fossils\" Diener, 2016 Figure 2 . From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides.",
"From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as \"sponges\" for other regulatory RNAs. information in the form of hairpin-loops . .",
"information in the form of hairpin-loops . . They can generate copies of themselves in the appropriate environment. They were designated as the \"frontiers of life\" . . The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 Pennazio and Roggero, 2000 . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens.",
"Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid ASBVd . Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle. Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss.",
"Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle.",
"Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA CarSV RNA cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners?",
"This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation. The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs.",
"The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules . . Their structure is reminiscent of catalytically active ribozymes. There is an exceptional viroid that gained coding information and entered the human liver Taylor, 2009 . The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides.",
"The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly.",
"Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus HBV , which delivers the coat Taylor, 2009 Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist? In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct.",
"In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as \"error catastrophe, \" which prevents survival Eigen, 1971 Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions.",
"The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma Crick, 1968 . Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions.",
"Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world.",
"Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed \"virus-first, \" such as \"protein-first\", \"metabolism-fist\" or the \"lipid world\" Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012 . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing . . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA.",
". Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert Crick, 1968; Gilbert, 1986 . What a vision! Our world was almost 50 years later defined as \"RNAprotein\" world Altman, 2013 . One can speculate our world was built of ribozymes or viroids, which means \"viruses first.\" ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today.",
"ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase.",
"Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 !",
"This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 .",
"The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity. The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.",
"The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again. These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out .",
"This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out . . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV Taylor, 2009 . An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth.",
"Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals core sequences as a stable composition Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 .",
"Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment. The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . .",
"Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 .",
"The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 . To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.",
"Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions. The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes.",
"Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism.",
"They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information Nasir and Caetano-Anolles, 2015 . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer HGT La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018 . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes.",
"A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million mio basepairs Mitch, 2017 . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia . . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp .",
". The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp . Figure 3 . The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses.",
"They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive La Scola et al., 2008; Fischer and Hackl, 2016 . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 .",
"The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 . Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter . . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today.",
". The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria see below . Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990.",
"Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful?",
"It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants Gibson et al., 2008 Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans.",
"His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt . . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name . . The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA.",
". The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length Figure 3 . The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses!",
"There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence .",
"Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence . . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses ERVs and related retroelements REs . . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome.",
"In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV HERV elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats LTR that act as promoters . . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence Temin, 1985 . The actual number is about 45% as estimated today . .",
"The actual number is about 45% as estimated today . . In some genes such as the Protein Kinase Inhibitor B PKIB gene we determined about 70% retrovirusrelated sequences Moelling and Broecker, 2015 . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago MYA Hayward, 2017 . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp.",
"Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 .",
"It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 . A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses.",
"They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses? These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 .",
"REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017 . This gene duplication dates back to 500 mio years Cotton and Page, 2005 . Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 .",
"Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred. Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer .",
"Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer . . HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name.",
"The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as \"necessary factor\" Flint, 2015 . Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally.",
"Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections . . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose.",
"This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described Galvani and Slatkin, 2003 . Viruses have been proven to be drivers of evolution Villarreal and Witzany, 2010 , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . .",
"In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity Villarreal, 2009 . Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 .",
"Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity.",
"The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus GALV . Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations.",
"Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus JSRV , whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by \"superinfection exclusion\" Tarlinton, 2012 . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . .",
"The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . . Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors Villarreal, 2011 . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity Charpentier and Doudna, 2013 . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle.",
"It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as \"superinfection exclusion\" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections.",
"This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text. to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 .",
"to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of \"endogenization\" of incoming genetic material for subsequent exclusion. One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations?",
"There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations? The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection.",
"The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by \"downstream promotion\" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer . . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless.",
"As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon Broecker et al., 2016b . As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency .",
"As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency . . Thus, the role of ERVs may be more complex than we presently know. Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are \"locked\" inside the cells and are major drivers of the increase of genetic complexity Cordaux and Batzer, 2009 . One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . .",
"One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease Beltz, 2018 . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection Katzourakis and Gifford, 2010 . There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 .",
"This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 . An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago BYA Embley and Martin, 2006 . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet.",
"Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii the causative agent of Q fever , Mycobacterium leprae, M. tuberculosis, and M. mycoides . . The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes.",
"Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life.",
"Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution . . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts. Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells?",
"Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic? Viruses are minimalistic in their composition and must have undergone stringent gene reductions Flint, 2015 . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information.",
"Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . .",
"Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . . These oncogenes were in most cases taken up by the retroviruses at the expense of structural gag , replicating pol or envelope env genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells.",
"They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src for sarcoma oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome Broecker et al., 2016a . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes. There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis .",
"There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis . . Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants.",
"In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species. Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution.",
"Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 .",
"Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex.",
"By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication Moelling, 2012 Moelling, , 2013 . Therefore, the question may be repeated: \"Are viruses our oldest ancestors?\" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong.",
"Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled \"Viruses: More Friends Than Foes\" Moelling, 2017 . The scenario described here focuses on viruses as drivers of evolution. The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . .",
"Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life. The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence."
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Does RNA replication need polymerase enzymes? | Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. | [
"The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution?",
"Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes.",
"Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life.",
"Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.” Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes . . Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids.",
". Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules aptamers , 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds.",
"The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube Lincoln and Joyce, 2009 . Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity.",
"Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid PNA , with more stable peptide bonds instead of phosphodiester bonds Zhang and Cech, 1997; Joyce, 2002 . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides Wilson and Szostak, 1999 . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes.",
"Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs Orgel, 2004 . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids.",
"They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis Meierhenrich, 2008 . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started . . There, temperature gradients and clay containing minerals such as magnesium or manganese are available.",
". There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication . . tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code.",
". tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: \"The ribosome is a ribozyme\" Cech, 2000 , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 .",
"Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 . Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro . . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV . . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . .",
". Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases . . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes Harish and Caetano-Anolles, 2012 . These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase.",
"Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme a reductase as today, and allow for DNA polymerization Wilson and Szostak, 1999; Joyce, 2002 . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin Moelling, 2012 Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . .",
"tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins Moelling and Broecker, 2015; Moelling et al., 2017 Figure 1 . It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the \"Encyclopedia of DNA Elements\" ENCODE project. ncDNA amounts to more than 98% of the human DNA genome . .",
"ncDNA amounts to more than 98% of the human DNA genome . . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression.",
"ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information.",
"DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 .",
"Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 . The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat . . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?",
"What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners? According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it \"forgotten\" to be removed? Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011 . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA.",
"Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus Figure 1 ? Patel et al. . demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 .",
"demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions Simon and Zimmerly, 2008; Lescot et al., 2016 . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world.",
"It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H RNase H is an essential enzyme of retroviruses . . The RNase H turned out to be one of the five most frequent and ancient proteins Ma et al., 2008 that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions . . Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 .",
". Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus TMV , and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 .",
"The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 . They look like \"frozen\" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element. Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 .",
"Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as \"living fossils\" Diener, 2016 Figure 2 . From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides.",
"From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as \"sponges\" for other regulatory RNAs. information in the form of hairpin-loops . .",
"information in the form of hairpin-loops . . They can generate copies of themselves in the appropriate environment. They were designated as the \"frontiers of life\" . . The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 Pennazio and Roggero, 2000 . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens.",
"Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid ASBVd . Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle. Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss.",
"Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle.",
"Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA CarSV RNA cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners?",
"This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation. The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs.",
"The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules . . Their structure is reminiscent of catalytically active ribozymes. There is an exceptional viroid that gained coding information and entered the human liver Taylor, 2009 . The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides.",
"The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly.",
"Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus HBV , which delivers the coat Taylor, 2009 Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist? In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct.",
"In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as \"error catastrophe, \" which prevents survival Eigen, 1971 Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions.",
"The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma Crick, 1968 . Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions.",
"Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world.",
"Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed \"virus-first, \" such as \"protein-first\", \"metabolism-fist\" or the \"lipid world\" Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012 . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing . . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA.",
". Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert Crick, 1968; Gilbert, 1986 . What a vision! Our world was almost 50 years later defined as \"RNAprotein\" world Altman, 2013 . One can speculate our world was built of ribozymes or viroids, which means \"viruses first.\" ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today.",
"ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase.",
"Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 !",
"This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 .",
"The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity. The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.",
"The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again. These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out .",
"This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out . . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV Taylor, 2009 . An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth.",
"Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals core sequences as a stable composition Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 .",
"Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment. The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . .",
"Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 .",
"The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 . To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.",
"Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions. The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes.",
"Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism.",
"They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information Nasir and Caetano-Anolles, 2015 . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer HGT La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018 . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes.",
"A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million mio basepairs Mitch, 2017 . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia . . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp .",
". The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp . Figure 3 . The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses.",
"They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive La Scola et al., 2008; Fischer and Hackl, 2016 . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 .",
"The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 . Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter . . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today.",
". The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria see below . Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990.",
"Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful?",
"It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants Gibson et al., 2008 Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans.",
"His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt . . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name . . The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA.",
". The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length Figure 3 . The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses!",
"There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence .",
"Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence . . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses ERVs and related retroelements REs . . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome.",
"In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV HERV elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats LTR that act as promoters . . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence Temin, 1985 . The actual number is about 45% as estimated today . .",
"The actual number is about 45% as estimated today . . In some genes such as the Protein Kinase Inhibitor B PKIB gene we determined about 70% retrovirusrelated sequences Moelling and Broecker, 2015 . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago MYA Hayward, 2017 . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp.",
"Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 .",
"It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 . A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses.",
"They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses? These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 .",
"REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017 . This gene duplication dates back to 500 mio years Cotton and Page, 2005 . Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 .",
"Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred. Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer .",
"Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer . . HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name.",
"The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as \"necessary factor\" Flint, 2015 . Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally.",
"Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections . . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose.",
"This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described Galvani and Slatkin, 2003 . Viruses have been proven to be drivers of evolution Villarreal and Witzany, 2010 , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . .",
"In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity Villarreal, 2009 . Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 .",
"Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity.",
"The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus GALV . Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations.",
"Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus JSRV , whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by \"superinfection exclusion\" Tarlinton, 2012 . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . .",
"The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . . Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors Villarreal, 2011 . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity Charpentier and Doudna, 2013 . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle.",
"It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as \"superinfection exclusion\" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections.",
"This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text. to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 .",
"to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of \"endogenization\" of incoming genetic material for subsequent exclusion. One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations?",
"There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations? The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection.",
"The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by \"downstream promotion\" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer . . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless.",
"As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon Broecker et al., 2016b . As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency .",
"As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency . . Thus, the role of ERVs may be more complex than we presently know. Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are \"locked\" inside the cells and are major drivers of the increase of genetic complexity Cordaux and Batzer, 2009 . One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . .",
"One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease Beltz, 2018 . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection Katzourakis and Gifford, 2010 . There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 .",
"This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 . An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago BYA Embley and Martin, 2006 . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet.",
"Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii the causative agent of Q fever , Mycobacterium leprae, M. tuberculosis, and M. mycoides . . The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes.",
"Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life.",
"Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution . . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts. Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells?",
"Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic? Viruses are minimalistic in their composition and must have undergone stringent gene reductions Flint, 2015 . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information.",
"Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . .",
"Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . . These oncogenes were in most cases taken up by the retroviruses at the expense of structural gag , replicating pol or envelope env genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells.",
"They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src for sarcoma oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome Broecker et al., 2016a . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes. There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis .",
"There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis . . Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants.",
"In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species. Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution.",
"Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 .",
"Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex.",
"By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication Moelling, 2012 Moelling, , 2013 . Therefore, the question may be repeated: \"Are viruses our oldest ancestors?\" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong.",
"Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled \"Viruses: More Friends Than Foes\" Moelling, 2017 . The scenario described here focuses on viruses as drivers of evolution. The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . .",
"Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life. The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence."
] | 1,690 | 1,182 |
How can DNA arise chemically from RNA? | deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. | [
"The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution?",
"Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes.",
"Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life.",
"Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.” Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes . . Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids.",
". Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules aptamers , 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds.",
"The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube Lincoln and Joyce, 2009 . Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity.",
"Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid PNA , with more stable peptide bonds instead of phosphodiester bonds Zhang and Cech, 1997; Joyce, 2002 . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides Wilson and Szostak, 1999 . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes.",
"Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs Orgel, 2004 . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids.",
"They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis Meierhenrich, 2008 . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started . . There, temperature gradients and clay containing minerals such as magnesium or manganese are available.",
". There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication . . tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code.",
". tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: \"The ribosome is a ribozyme\" Cech, 2000 , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 .",
"Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 . Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro . . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV . . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . .",
". Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases . . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes Harish and Caetano-Anolles, 2012 . These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase.",
"Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme a reductase as today, and allow for DNA polymerization Wilson and Szostak, 1999; Joyce, 2002 . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin Moelling, 2012 Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . .",
"tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins Moelling and Broecker, 2015; Moelling et al., 2017 Figure 1 . It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the \"Encyclopedia of DNA Elements\" ENCODE project. ncDNA amounts to more than 98% of the human DNA genome . .",
"ncDNA amounts to more than 98% of the human DNA genome . . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression.",
"ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information.",
"DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 .",
"Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 . The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat . . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?",
"What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners? According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it \"forgotten\" to be removed? Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011 . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA.",
"Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus Figure 1 ? Patel et al. . demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 .",
"demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions Simon and Zimmerly, 2008; Lescot et al., 2016 . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world.",
"It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H RNase H is an essential enzyme of retroviruses . . The RNase H turned out to be one of the five most frequent and ancient proteins Ma et al., 2008 that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions . . Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 .",
". Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus TMV , and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 .",
"The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 . They look like \"frozen\" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element. Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 .",
"Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as \"living fossils\" Diener, 2016 Figure 2 . From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides.",
"From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as \"sponges\" for other regulatory RNAs. information in the form of hairpin-loops . .",
"information in the form of hairpin-loops . . They can generate copies of themselves in the appropriate environment. They were designated as the \"frontiers of life\" . . The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 Pennazio and Roggero, 2000 . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens.",
"Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid ASBVd . Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle. Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss.",
"Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle.",
"Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA CarSV RNA cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners?",
"This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation. The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs.",
"The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules . . Their structure is reminiscent of catalytically active ribozymes. There is an exceptional viroid that gained coding information and entered the human liver Taylor, 2009 . The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides.",
"The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly.",
"Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus HBV , which delivers the coat Taylor, 2009 Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist? In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct.",
"In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as \"error catastrophe, \" which prevents survival Eigen, 1971 Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions.",
"The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma Crick, 1968 . Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions.",
"Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world.",
"Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed \"virus-first, \" such as \"protein-first\", \"metabolism-fist\" or the \"lipid world\" Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012 . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing . . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA.",
". Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert Crick, 1968; Gilbert, 1986 . What a vision! Our world was almost 50 years later defined as \"RNAprotein\" world Altman, 2013 . One can speculate our world was built of ribozymes or viroids, which means \"viruses first.\" ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today.",
"ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase.",
"Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 !",
"This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 .",
"The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity. The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.",
"The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again. These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out .",
"This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out . . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV Taylor, 2009 . An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth.",
"Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals core sequences as a stable composition Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 .",
"Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment. The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . .",
"Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 .",
"The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 . To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.",
"Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions. The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes.",
"Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism.",
"They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information Nasir and Caetano-Anolles, 2015 . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer HGT La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018 . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes.",
"A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million mio basepairs Mitch, 2017 . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia . . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp .",
". The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp . Figure 3 . The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses.",
"They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive La Scola et al., 2008; Fischer and Hackl, 2016 . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 .",
"The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 . Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter . . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today.",
". The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria see below . Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990.",
"Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful?",
"It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants Gibson et al., 2008 Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans.",
"His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt . . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name . . The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA.",
". The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length Figure 3 . The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses!",
"There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence .",
"Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence . . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses ERVs and related retroelements REs . . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome.",
"In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV HERV elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats LTR that act as promoters . . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence Temin, 1985 . The actual number is about 45% as estimated today . .",
"The actual number is about 45% as estimated today . . In some genes such as the Protein Kinase Inhibitor B PKIB gene we determined about 70% retrovirusrelated sequences Moelling and Broecker, 2015 . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago MYA Hayward, 2017 . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp.",
"Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 .",
"It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 . A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses.",
"They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses? These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 .",
"REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017 . This gene duplication dates back to 500 mio years Cotton and Page, 2005 . Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 .",
"Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred. Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer .",
"Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer . . HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name.",
"The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as \"necessary factor\" Flint, 2015 . Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally.",
"Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections . . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose.",
"This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described Galvani and Slatkin, 2003 . Viruses have been proven to be drivers of evolution Villarreal and Witzany, 2010 , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . .",
"In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity Villarreal, 2009 . Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 .",
"Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity.",
"The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus GALV . Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations.",
"Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus JSRV , whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by \"superinfection exclusion\" Tarlinton, 2012 . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . .",
"The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . . Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors Villarreal, 2011 . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity Charpentier and Doudna, 2013 . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle.",
"It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as \"superinfection exclusion\" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections.",
"This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text. to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 .",
"to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of \"endogenization\" of incoming genetic material for subsequent exclusion. One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations?",
"There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations? The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection.",
"The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by \"downstream promotion\" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer . . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless.",
"As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon Broecker et al., 2016b . As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency .",
"As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency . . Thus, the role of ERVs may be more complex than we presently know. Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are \"locked\" inside the cells and are major drivers of the increase of genetic complexity Cordaux and Batzer, 2009 . One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . .",
"One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease Beltz, 2018 . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection Katzourakis and Gifford, 2010 . There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 .",
"This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 . An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago BYA Embley and Martin, 2006 . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet.",
"Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii the causative agent of Q fever , Mycobacterium leprae, M. tuberculosis, and M. mycoides . . The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes.",
"Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life.",
"Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution . . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts. Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells?",
"Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic? Viruses are minimalistic in their composition and must have undergone stringent gene reductions Flint, 2015 . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information.",
"Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . .",
"Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . . These oncogenes were in most cases taken up by the retroviruses at the expense of structural gag , replicating pol or envelope env genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells.",
"They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src for sarcoma oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome Broecker et al., 2016a . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes. There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis .",
"There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis . . Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants.",
"In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species. Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution.",
"Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 .",
"Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex.",
"By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication Moelling, 2012 Moelling, , 2013 . Therefore, the question may be repeated: \"Are viruses our oldest ancestors?\" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong.",
"Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled \"Viruses: More Friends Than Foes\" Moelling, 2017 . The scenario described here focuses on viruses as drivers of evolution. The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . .",
"Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life. The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence."
] | 1,690 | 1,183 |
What do ribozymes consist of? | circular single-stranded RNAs | [
"The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution?",
"Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes.",
"Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life.",
"Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.” Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes . . Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids.",
". Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules aptamers , 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds.",
"The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube Lincoln and Joyce, 2009 . Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity.",
"Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid PNA , with more stable peptide bonds instead of phosphodiester bonds Zhang and Cech, 1997; Joyce, 2002 . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides Wilson and Szostak, 1999 . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes.",
"Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs Orgel, 2004 . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids.",
"They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis Meierhenrich, 2008 . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started . . There, temperature gradients and clay containing minerals such as magnesium or manganese are available.",
". There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication . . tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code.",
". tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: \"The ribosome is a ribozyme\" Cech, 2000 , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 .",
"Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 . Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro . . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV . . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . .",
". Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases . . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes Harish and Caetano-Anolles, 2012 . These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase.",
"Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme a reductase as today, and allow for DNA polymerization Wilson and Szostak, 1999; Joyce, 2002 . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin Moelling, 2012 Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . .",
"tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins Moelling and Broecker, 2015; Moelling et al., 2017 Figure 1 . It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the \"Encyclopedia of DNA Elements\" ENCODE project. ncDNA amounts to more than 98% of the human DNA genome . .",
"ncDNA amounts to more than 98% of the human DNA genome . . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression.",
"ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information.",
"DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 .",
"Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 . The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat . . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?",
"What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners? According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it \"forgotten\" to be removed? Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011 . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA.",
"Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus Figure 1 ? Patel et al. . demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 .",
"demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions Simon and Zimmerly, 2008; Lescot et al., 2016 . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world.",
"It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H RNase H is an essential enzyme of retroviruses . . The RNase H turned out to be one of the five most frequent and ancient proteins Ma et al., 2008 that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions . . Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 .",
". Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus TMV , and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 .",
"The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 . They look like \"frozen\" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element. Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 .",
"Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as \"living fossils\" Diener, 2016 Figure 2 . From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides.",
"From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as \"sponges\" for other regulatory RNAs. information in the form of hairpin-loops . .",
"information in the form of hairpin-loops . . They can generate copies of themselves in the appropriate environment. They were designated as the \"frontiers of life\" . . The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 Pennazio and Roggero, 2000 . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens.",
"Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid ASBVd . Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle. Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss.",
"Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle.",
"Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA CarSV RNA cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners?",
"This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation. The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs.",
"The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules . . Their structure is reminiscent of catalytically active ribozymes. There is an exceptional viroid that gained coding information and entered the human liver Taylor, 2009 . The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides.",
"The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly.",
"Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus HBV , which delivers the coat Taylor, 2009 Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist? In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct.",
"In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as \"error catastrophe, \" which prevents survival Eigen, 1971 Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions.",
"The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma Crick, 1968 . Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions.",
"Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world.",
"Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed \"virus-first, \" such as \"protein-first\", \"metabolism-fist\" or the \"lipid world\" Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012 . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing . . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA.",
". Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert Crick, 1968; Gilbert, 1986 . What a vision! Our world was almost 50 years later defined as \"RNAprotein\" world Altman, 2013 . One can speculate our world was built of ribozymes or viroids, which means \"viruses first.\" ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today.",
"ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase.",
"Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 !",
"This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 .",
"The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity. The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.",
"The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again. These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out .",
"This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out . . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV Taylor, 2009 . An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth.",
"Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals core sequences as a stable composition Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 .",
"Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment. The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . .",
"Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 .",
"The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 . To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.",
"Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions. The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes.",
"Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism.",
"They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information Nasir and Caetano-Anolles, 2015 . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer HGT La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018 . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes.",
"A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million mio basepairs Mitch, 2017 . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia . . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp .",
". The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp . Figure 3 . The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses.",
"They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive La Scola et al., 2008; Fischer and Hackl, 2016 . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 .",
"The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 . Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter . . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today.",
". The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria see below . Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990.",
"Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful?",
"It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants Gibson et al., 2008 Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans.",
"His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt . . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name . . The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA.",
". The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length Figure 3 . The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses!",
"There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence .",
"Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence . . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses ERVs and related retroelements REs . . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome.",
"In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV HERV elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats LTR that act as promoters . . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence Temin, 1985 . The actual number is about 45% as estimated today . .",
"The actual number is about 45% as estimated today . . In some genes such as the Protein Kinase Inhibitor B PKIB gene we determined about 70% retrovirusrelated sequences Moelling and Broecker, 2015 . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago MYA Hayward, 2017 . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp.",
"Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 .",
"It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 . A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses.",
"They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses? These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 .",
"REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017 . This gene duplication dates back to 500 mio years Cotton and Page, 2005 . Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 .",
"Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred. Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer .",
"Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer . . HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name.",
"The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as \"necessary factor\" Flint, 2015 . Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally.",
"Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections . . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose.",
"This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described Galvani and Slatkin, 2003 . Viruses have been proven to be drivers of evolution Villarreal and Witzany, 2010 , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . .",
"In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity Villarreal, 2009 . Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 .",
"Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity.",
"The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus GALV . Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations.",
"Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus JSRV , whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by \"superinfection exclusion\" Tarlinton, 2012 . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . .",
"The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . . Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors Villarreal, 2011 . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity Charpentier and Doudna, 2013 . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle.",
"It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as \"superinfection exclusion\" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections.",
"This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text. to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 .",
"to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of \"endogenization\" of incoming genetic material for subsequent exclusion. One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations?",
"There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations? The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection.",
"The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by \"downstream promotion\" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer . . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless.",
"As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon Broecker et al., 2016b . As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency .",
"As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency . . Thus, the role of ERVs may be more complex than we presently know. Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are \"locked\" inside the cells and are major drivers of the increase of genetic complexity Cordaux and Batzer, 2009 . One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . .",
"One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease Beltz, 2018 . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection Katzourakis and Gifford, 2010 . There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 .",
"This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 . An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago BYA Embley and Martin, 2006 . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet.",
"Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii the causative agent of Q fever , Mycobacterium leprae, M. tuberculosis, and M. mycoides . . The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes.",
"Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life.",
"Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution . . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts. Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells?",
"Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic? Viruses are minimalistic in their composition and must have undergone stringent gene reductions Flint, 2015 . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information.",
"Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . .",
"Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . . These oncogenes were in most cases taken up by the retroviruses at the expense of structural gag , replicating pol or envelope env genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells.",
"They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src for sarcoma oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome Broecker et al., 2016a . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes. There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis .",
"There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis . . Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants.",
"In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species. Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution.",
"Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 .",
"Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex.",
"By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication Moelling, 2012 Moelling, , 2013 . Therefore, the question may be repeated: \"Are viruses our oldest ancestors?\" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong.",
"Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled \"Viruses: More Friends Than Foes\" Moelling, 2017 . The scenario described here focuses on viruses as drivers of evolution. The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . .",
"Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life. The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence."
] | 1,690 | 1,184 |
What do ribozymes lack? | the genetic triplet code and do not encode proteins | [
"The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution?",
"Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes.",
"Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life.",
"Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.” Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes . . Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids.",
". Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules aptamers , 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds.",
"The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube Lincoln and Joyce, 2009 . Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity.",
"Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid PNA , with more stable peptide bonds instead of phosphodiester bonds Zhang and Cech, 1997; Joyce, 2002 . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides Wilson and Szostak, 1999 . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes.",
"Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs Orgel, 2004 . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids.",
"They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis Meierhenrich, 2008 . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started . . There, temperature gradients and clay containing minerals such as magnesium or manganese are available.",
". There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication . . tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code.",
". tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: \"The ribosome is a ribozyme\" Cech, 2000 , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 .",
"Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 . Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro . . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV . . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . .",
". Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases . . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes Harish and Caetano-Anolles, 2012 . These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase.",
"Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme a reductase as today, and allow for DNA polymerization Wilson and Szostak, 1999; Joyce, 2002 . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin Moelling, 2012 Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . .",
"tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins Moelling and Broecker, 2015; Moelling et al., 2017 Figure 1 . It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the \"Encyclopedia of DNA Elements\" ENCODE project. ncDNA amounts to more than 98% of the human DNA genome . .",
"ncDNA amounts to more than 98% of the human DNA genome . . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression.",
"ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information.",
"DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 .",
"Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 . The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat . . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?",
"What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners? According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it \"forgotten\" to be removed? Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011 . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA.",
"Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus Figure 1 ? Patel et al. . demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 .",
"demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions Simon and Zimmerly, 2008; Lescot et al., 2016 . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world.",
"It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H RNase H is an essential enzyme of retroviruses . . The RNase H turned out to be one of the five most frequent and ancient proteins Ma et al., 2008 that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions . . Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 .",
". Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus TMV , and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 .",
"The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 . They look like \"frozen\" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element. Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 .",
"Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as \"living fossils\" Diener, 2016 Figure 2 . From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides.",
"From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as \"sponges\" for other regulatory RNAs. information in the form of hairpin-loops . .",
"information in the form of hairpin-loops . . They can generate copies of themselves in the appropriate environment. They were designated as the \"frontiers of life\" . . The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 Pennazio and Roggero, 2000 . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens.",
"Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid ASBVd . Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle. Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss.",
"Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle.",
"Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA CarSV RNA cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners?",
"This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation. The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs.",
"The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules . . Their structure is reminiscent of catalytically active ribozymes. There is an exceptional viroid that gained coding information and entered the human liver Taylor, 2009 . The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides.",
"The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly.",
"Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus HBV , which delivers the coat Taylor, 2009 Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist? In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct.",
"In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as \"error catastrophe, \" which prevents survival Eigen, 1971 Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions.",
"The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma Crick, 1968 . Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions.",
"Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world.",
"Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed \"virus-first, \" such as \"protein-first\", \"metabolism-fist\" or the \"lipid world\" Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012 . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing . . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA.",
". Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert Crick, 1968; Gilbert, 1986 . What a vision! Our world was almost 50 years later defined as \"RNAprotein\" world Altman, 2013 . One can speculate our world was built of ribozymes or viroids, which means \"viruses first.\" ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today.",
"ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase.",
"Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 !",
"This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 .",
"The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity. The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.",
"The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again. These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out .",
"This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out . . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV Taylor, 2009 . An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth.",
"Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals core sequences as a stable composition Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 .",
"Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment. The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . .",
"Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 .",
"The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 . To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.",
"Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions. The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes.",
"Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism.",
"They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information Nasir and Caetano-Anolles, 2015 . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer HGT La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018 . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes.",
"A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million mio basepairs Mitch, 2017 . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia . . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp .",
". The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp . Figure 3 . The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses.",
"They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive La Scola et al., 2008; Fischer and Hackl, 2016 . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 .",
"The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 . Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter . . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today.",
". The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria see below . Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990.",
"Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful?",
"It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants Gibson et al., 2008 Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans.",
"His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt . . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name . . The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA.",
". The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length Figure 3 . The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses!",
"There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence .",
"Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence . . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses ERVs and related retroelements REs . . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome.",
"In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV HERV elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats LTR that act as promoters . . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence Temin, 1985 . The actual number is about 45% as estimated today . .",
"The actual number is about 45% as estimated today . . In some genes such as the Protein Kinase Inhibitor B PKIB gene we determined about 70% retrovirusrelated sequences Moelling and Broecker, 2015 . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago MYA Hayward, 2017 . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp.",
"Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 .",
"It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 . A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses.",
"They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses? These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 .",
"REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017 . This gene duplication dates back to 500 mio years Cotton and Page, 2005 . Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 .",
"Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred. Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer .",
"Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer . . HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name.",
"The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as \"necessary factor\" Flint, 2015 . Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally.",
"Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections . . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose.",
"This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described Galvani and Slatkin, 2003 . Viruses have been proven to be drivers of evolution Villarreal and Witzany, 2010 , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . .",
"In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity Villarreal, 2009 . Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 .",
"Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity.",
"The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus GALV . Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations.",
"Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus JSRV , whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by \"superinfection exclusion\" Tarlinton, 2012 . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . .",
"The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . . Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors Villarreal, 2011 . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity Charpentier and Doudna, 2013 . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle.",
"It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as \"superinfection exclusion\" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections.",
"This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text. to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 .",
"to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of \"endogenization\" of incoming genetic material for subsequent exclusion. One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations?",
"There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations? The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection.",
"The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by \"downstream promotion\" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer . . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless.",
"As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon Broecker et al., 2016b . As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency .",
"As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency . . Thus, the role of ERVs may be more complex than we presently know. Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are \"locked\" inside the cells and are major drivers of the increase of genetic complexity Cordaux and Batzer, 2009 . One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . .",
"One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease Beltz, 2018 . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection Katzourakis and Gifford, 2010 . There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 .",
"This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 . An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago BYA Embley and Martin, 2006 . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet.",
"Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii the causative agent of Q fever , Mycobacterium leprae, M. tuberculosis, and M. mycoides . . The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes.",
"Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life.",
"Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution . . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts. Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells?",
"Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic? Viruses are minimalistic in their composition and must have undergone stringent gene reductions Flint, 2015 . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information.",
"Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . .",
"Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . . These oncogenes were in most cases taken up by the retroviruses at the expense of structural gag , replicating pol or envelope env genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells.",
"They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src for sarcoma oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome Broecker et al., 2016a . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes. There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis .",
"There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis . . Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants.",
"In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species. Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution.",
"Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 .",
"Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex.",
"By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication Moelling, 2012 Moelling, , 2013 . Therefore, the question may be repeated: \"Are viruses our oldest ancestors?\" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong.",
"Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled \"Viruses: More Friends Than Foes\" Moelling, 2017 . The scenario described here focuses on viruses as drivers of evolution. The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . .",
"Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life. The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence."
] | 1,690 | 1,185 |
What do ribozymes exhibit? | structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. | [
"The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution?",
"Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes.",
"Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life.",
"Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.” Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes . . Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids.",
". Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules aptamers , 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds.",
"The sequence space for 220-mer RNAs is about 3 × 10 132 Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006 . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube Lincoln and Joyce, 2009 . Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity.",
"Ribozymes can form peptide bonds between amino acids Zhang and Cech, 1997 . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid PNA , with more stable peptide bonds instead of phosphodiester bonds Zhang and Cech, 1997; Joyce, 2002 . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides Wilson and Szostak, 1999 . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes.",
"Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA ncRNA before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs Orgel, 2004 . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids.",
"They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis Meierhenrich, 2008 . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started . . There, temperature gradients and clay containing minerals such as magnesium or manganese are available.",
". There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication . . tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code.",
". tRNA-amino acid complexes can find RNAs as \"mRNAs.\" Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: \"The ribosome is a ribozyme\" Cech, 2000 , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 .",
"Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe Meierhenrich, 2008 . Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro . . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV . . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . .",
". Peptides can enhance the catalytic activity of ribozymes up to a 100-fold . . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases . . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes Harish and Caetano-Anolles, 2012 . These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase.",
"Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme a reductase as today, and allow for DNA polymerization Wilson and Szostak, 1999; Joyce, 2002 . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin Moelling, 2012 Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . .",
"tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs . . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins Moelling and Broecker, 2015; Moelling et al., 2017 Figure 1 . It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the \"Encyclopedia of DNA Elements\" ENCODE project. ncDNA amounts to more than 98% of the human DNA genome . .",
"ncDNA amounts to more than 98% of the human DNA genome . . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression.",
"ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element TAR , the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information.",
"DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA ncRNA , ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 .",
"Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA Flint, 2015 . The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat . . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners?",
"What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners? According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it \"forgotten\" to be removed? Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011 . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA.",
"Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus Figure 1 ? Patel et al. . demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 .",
"demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a \"one pot\" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules Miller, 1953; Miller and Urey, 1959 . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions Simon and Zimmerly, 2008; Lescot et al., 2016 . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world.",
"It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the \"real\" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H RNase H is an essential enzyme of retroviruses . . The RNase H turned out to be one of the five most frequent and ancient proteins Ma et al., 2008 that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions . . Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 .",
". Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures TLS , resembling these early RNAs Dreher, 2009 . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus TMV , and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 .",
"The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as \"mimicry, \" enhancing translation Dreher, 2009 Dreher, , 2010 . They look like \"frozen\" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element. Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 .",
"Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses Chela-Flores, 1994 . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as \"living fossils\" Diener, 2016 Figure 2 . From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides.",
"From infected potatoes, Diener isolated the Potato spindle tuber viroid PSTVd whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as \"sponges\" for other regulatory RNAs. information in the form of hairpin-loops . .",
"information in the form of hairpin-loops . . They can generate copies of themselves in the appropriate environment. They were designated as the \"frontiers of life\" . . The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 Pennazio and Roggero, 2000 . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens.",
"Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid ASBVd . Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle. Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss.",
"Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle.",
"Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA CarSV RNA cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners?",
"This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers Flores et al., 2005 Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation. The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs.",
"The circular RNA circRNA is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a \"sponge\" absorbing small RNAs. Micro RNAs miRNAs are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules . . Their structure is reminiscent of catalytically active ribozymes. There is an exceptional viroid that gained coding information and entered the human liver Taylor, 2009 . The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides.",
"The viroid is known as hepatitis delta virus HDV . It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly.",
"Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen HDVAg that occurs in two forms, the small-HDVAg 24 kDa supporting replication and the large-HDVAg 27 kDa that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus HBV , which delivers the coat Taylor, 2009 Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist? In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct.",
"In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets .. Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as \"error catastrophe, \" which prevents survival Eigen, 1971 Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions.",
"The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma Crick, 1968 . Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions.",
"Even an information flow from protein to DNA has been described for some archaeal proteins . . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world.",
"Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed \"virus-first, \" such as \"protein-first\", \"metabolism-fist\" or the \"lipid world\" Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012 . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing . . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA.",
". Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert Crick, 1968; Gilbert, 1986 . What a vision! Our world was almost 50 years later defined as \"RNAprotein\" world Altman, 2013 . One can speculate our world was built of ribozymes or viroids, which means \"viruses first.\" ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today.",
"ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase.",
"Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 !",
"This experiment performed in 1965 was designated as \"Spiegelman's Monster.\" Coding RNA became replicating ncRNA Spiegelman et al., 1965; Kacian et al., 1972 ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 .",
"The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population Sumper and Luce, 1975; Eigen, 2013 . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity. The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again.",
"The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again. These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out .",
"This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out . . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV Taylor, 2009 . An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth.",
"Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals core sequences as a stable composition Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 .",
"Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert Cotillard et al., 2013; Le Chatelier et al., 2013 . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment. The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . .",
"Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria . . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 .",
"The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes Segata, 2015 . To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions.",
"Deletions resulted in smaller genomes with reduced or absence of DNA repair genes . . Gene loss conferred a higher fitness to the bacteria under these experimental conditions. The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes.",
"Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae Emiliania huxleyi , but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism.",
"They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information Nasir and Caetano-Anolles, 2015 . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer HGT La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018 . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes.",
"A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds based on Holmes, 2011 Figure 3 . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million mio basepairs Mitch, 2017 . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia . . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp .",
". The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp . Figure 3 . The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses.",
"They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive La Scola et al., 2008; Fischer and Hackl, 2016 . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 .",
"The giant viruses may be considered as missing link between the two, because they harbor \"almost\" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline Figure 3 . Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter . . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today.",
". The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria see below . Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990.",
"Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria Giovannoni, 2017 , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful?",
"It harbors podophage-type phages, designated as \"pelagiphage\" . . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants Gibson et al., 2008 Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans.",
"His group synthesized a genome of 531,000 bp with 473 genes, 149 of them 32% with unknown functions . . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt . . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name . . The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA.",
". The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length Figure 3 . The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses!",
"There are about 10 33 viruses on our planet and they are present in every single existing species Suttle, 2005 . There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence .",
"Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence . . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses ERVs and related retroelements REs . . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome.",
"In addition, DNA transposable elements TEs move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV HERV elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats LTR that act as promoters . . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence Temin, 1985 . The actual number is about 45% as estimated today . .",
"The actual number is about 45% as estimated today . . In some genes such as the Protein Kinase Inhibitor B PKIB gene we determined about 70% retrovirusrelated sequences Moelling and Broecker, 2015 . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago MYA Hayward, 2017 . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp.",
"Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 .",
"It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames Wolf and Koonin, 2013 . A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses.",
"They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses? These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 .",
"REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations Cotton and Page, 2005 . Such duplications constitute large amounts of mammalian genomes Zhang, 2003 . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017 . This gene duplication dates back to 500 mio years Cotton and Page, 2005 . Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 .",
"Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers Vogelstein and Kinzler, 2002 . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred. Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer .",
"Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer . . HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name.",
"The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as \"necessary factor\" Flint, 2015 . Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally.",
"Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids CCR5-32 , disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections . . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose.",
"This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described Galvani and Slatkin, 2003 . Viruses have been proven to be drivers of evolution Villarreal and Witzany, 2010 , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . .",
"In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system . . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity Villarreal, 2009 . Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 .",
"Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years Hayward, 2017 . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity.",
"The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis Dewannieux and Heidmann, 2013 . The koalas in Australia are currently undergoing endogenization of a retrovirus koala retrovirus, KoRV in \"real time\" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus GALV . Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations.",
"Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus JSRV , whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by \"superinfection exclusion\" Tarlinton, 2012 . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . .",
"The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells . . Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors Villarreal, 2011 . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity Charpentier and Doudna, 2013 . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle.",
"It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms Figure 4 . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as \"superinfection exclusion\" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections.",
"This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text. to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 .",
"to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus SIV in some monkey species may be explained by endogenization Li et al., 2017 Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of \"endogenization\" of incoming genetic material for subsequent exclusion. One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations?",
"There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome . . How long this may take is not known -10 generations? The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection.",
"The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome Lander et al., 2001; Cordaux and Batzer, 2009 . The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by \"downstream promotion\" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer . . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless.",
"As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon Broecker et al., 2016b . As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency .",
"As shown for the koalas, ERVs can confer immunity to viral infections Feschotte and Gilbert, 2012 . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency . . Thus, the role of ERVs may be more complex than we presently know. Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are \"locked\" inside the cells and are major drivers of the increase of genetic complexity Cordaux and Batzer, 2009 . One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . .",
"One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats . . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease Beltz, 2018 . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection Katzourakis and Gifford, 2010 . There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 .",
"This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria Margulis, 1970 Margulis, , 1993 . An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago BYA Embley and Martin, 2006 . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet.",
"Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii the causative agent of Q fever , Mycobacterium leprae, M. tuberculosis, and M. mycoides . . The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes.",
"Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life.",
"Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution . . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts. Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells?",
"Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic? Viruses are minimalistic in their composition and must have undergone stringent gene reductions Flint, 2015 . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information.",
"Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . .",
"Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs . . These oncogenes were in most cases taken up by the retroviruses at the expense of structural gag , replicating pol or envelope env genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells.",
"They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells Flint, 2015 . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src for sarcoma oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome Broecker et al., 2016a . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes. There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis .",
"There are indications that the uncontrolled activity of retro transposons in germline cells can result in diseases such as male infertility -presumably by \"error catastrophe, \" caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis . . Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants.",
"In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species. Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution.",
"Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 .",
"Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA Hayward, 2017 . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex.",
"By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication Moelling, 2012 Moelling, , 2013 . Therefore, the question may be repeated: \"Are viruses our oldest ancestors?\" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong.",
"Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled \"Viruses: More Friends Than Foes\" Moelling, 2017 . The scenario described here focuses on viruses as drivers of evolution. The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . .",
"Surprisingly, there are scientists who still believe in the \"pansperm hypothesis\" and think that retroviruses are of extraterrestric origin . . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life. The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence."
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What is the acronym SARS-CoV-2? | severe acute respiratory syndrome coronavirus 2 | [
"Identifying Locations with Possible Undetected Imported Severe Acute Respiratory Syndrome Coronavirus 2 Cases by Using Importation Predictions, Volume 26, Number 7—July 2020 Research Pablo Martinez De Salazar1Comments to Author , René Niehus, Aimee Taylor1, Caroline O’Flaherty Buckee, and Marc LipsitchComments to Author Author affiliations: Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA Suggested citation for this article Abstract Cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 infection exported from mainland China could lead to self-sustained outbreaks in other countries. By February 2020, several countries were reporting imported SARS-CoV-2 cases. To contain the virus, early detection of imported SARS-CoV-2 cases is critical. We used air travel volume estimates from Wuhan, China, to international destinations and a generalized linear regression model to identify locations that could have undetected imported cases. Our model can be adjusted to account for exportation of cases from other locations as the virus spreads and more information on importations and transmission becomes available.",
"We used air travel volume estimates from Wuhan, China, to international destinations and a generalized linear regression model to identify locations that could have undetected imported cases. Our model can be adjusted to account for exportation of cases from other locations as the virus spreads and more information on importations and transmission becomes available. Early detection and appropriate control measures can reduce the risk for transmission in all locations. A novel coronavirus, later named severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 , was identified in December 2019 in the city of Wuhan, capital of Hubei Province, China, where cases were first confirmed .. During December 2019–February 2020, the number of confirmed cases increased drastically. Model estimates suggested that >75,000 persons were infected by January 25, 2020, and the epidemic had a doubling time of ≈6 days .. By the end of January 2020, travel restrictions were implemented for Wuhan and neighboring cities. Nonetheless, the virus spread from Wuhan to other cities in China and outside the country.",
"Model estimates suggested that >75,000 persons were infected by January 25, 2020, and the epidemic had a doubling time of ≈6 days .. By the end of January 2020, travel restrictions were implemented for Wuhan and neighboring cities. Nonetheless, the virus spread from Wuhan to other cities in China and outside the country. By February 4, 2020, a total of 23 locations outside mainland China reported cases, 22 of which reported imported cases; Spain reported a case caused by secondary transmission .. Most cases imported to other locations have been linked to recent travel history from China ., suggesting that air travel plays a major role in exportation of cases to locations outside of China. To prevent other cities and countries from becoming epicenters of the SARS-CoV-2 epidemic, substantial targeted public health interventions are required to detect cases and control local spread of the virus. We collected estimates of air travel volume from Wuhan to 194 international destinations.",
"To prevent other cities and countries from becoming epicenters of the SARS-CoV-2 epidemic, substantial targeted public health interventions are required to detect cases and control local spread of the virus. We collected estimates of air travel volume from Wuhan to 194 international destinations. We then identified 49 countries that had a score of >49.2/100 on category 2, Early Detection and Reporting of Epidemics of Potential International Concern, of the Global Health Security GHS Index .. We assumed these locations would be proficient at detecting SARS-CoV-2 and reporting confirmed imported cases, which we refer to as imported-and-reported cases. We ran a generalized linear regression model on this subset; based on the results, we generated predictions for the remainder of the sample. Using these predictions, we identified locations that might not be detecting imported cases. Methods To identify locations reporting fewer than predicted imported SARS-CoV-2 infected cases, we fit a model to data from 49 locations outside mainland China with high surveillance capacity according to the GHS Index ..",
"Using these predictions, we identified locations that might not be detecting imported cases. Methods To identify locations reporting fewer than predicted imported SARS-CoV-2 infected cases, we fit a model to data from 49 locations outside mainland China with high surveillance capacity according to the GHS Index .. Among these, 17 had high travel connectivity to Wuhan and 32 have low connectivity to Wuhan. We considered locations to be countries without any position on territorial claims. We performed a Poisson regression by using the cumulative number of imported-and-reported SARS-CoV-2 cases in these 49 countries and the estimated number of daily airline passengers from the Wuhan airport. We then compared predictions from this model with imported-and-reported cases across 194 locations from the GHS Index, excluding China as the epicenter of the outbreak.",
"We performed a Poisson regression by using the cumulative number of imported-and-reported SARS-CoV-2 cases in these 49 countries and the estimated number of daily airline passengers from the Wuhan airport. We then compared predictions from this model with imported-and-reported cases across 194 locations from the GHS Index, excluding China as the epicenter of the outbreak. The model requires data on imported-and-reported cases of SARS-CoV-2 infection, daily air travel volume, and surveillance capacity. We obtained data on imported-and-reported cases aggregated by destination from the World Health Organization technical report issued February 4, 2020 .. We assumed a case count of 0 for locations not listed. We used February 4 as the cutoff for cumulative imported-and-reported case counts because exported cases from Hubei Province dropped rapidly after this date ., likely because of travel restrictions for the province implement on January 23. We defined imported-and-reported cases as those with known travel history from China; of those, 83% had a travel history from Hubei Province and 17% traveled from unknown locations in China .. We excluded reported cases likely caused by transmission outside of China or cases in which the transmission source was still under investigation ..",
"We used February 4 as the cutoff for cumulative imported-and-reported case counts because exported cases from Hubei Province dropped rapidly after this date ., likely because of travel restrictions for the province implement on January 23. We defined imported-and-reported cases as those with known travel history from China; of those, 83% had a travel history from Hubei Province and 17% traveled from unknown locations in China .. We excluded reported cases likely caused by transmission outside of China or cases in which the transmission source was still under investigation .. In addition, we excluded Hong Kong, Macau, and Taiwan from our model because locally transmitted and imported cases were not disaggregated in these locations. We obtained data on daily air travel from a network-based modeling study S. Lai et al., unpub. data, Link that reported monthly air travel volume estimates for the 27 locations outside mainland China that are most connected to Wuhan. These estimates were calculated from International Air Travel Association data from February 2018, which includes direct and indirect flight itineraries from Wuhan.",
"data, Link that reported monthly air travel volume estimates for the 27 locations outside mainland China that are most connected to Wuhan. These estimates were calculated from International Air Travel Association data from February 2018, which includes direct and indirect flight itineraries from Wuhan. For these 27 locations, estimated air travel volumes are >6 passengers/day. We assumed that travel volumes for locations not among the most connected are censored by a detection limit. We used a common method of dealing with censored data from environmental sampling ., or metabolomics ., to set the daily air travel volume to half the minimum previously reported. Therefore, we used 3 passengers/day for estimated travel volumes for the 167 locations from the GHS Index not listed by Lai et al.",
"We used a common method of dealing with censored data from environmental sampling ., or metabolomics ., to set the daily air travel volume to half the minimum previously reported. Therefore, we used 3 passengers/day for estimated travel volumes for the 167 locations from the GHS Index not listed by Lai et al. We tested the robustness of our results by using a set of alternative values of 0.1, 1, and 6 passengers/day for the censored data. We defined high surveillance locations as those with a GHS Index for category 2 above the 75th quantile. We assessed the number of high surveillance locations, those with 0 imported-and-reported cases, and low surveillance locations, those with case counts >1 Table . For our model, we assumed that the cumulative imported-and-reported case counts across 49 high surveillance locations follow a Poisson distribution from the beginning of the epidemic until February 4, 2020.",
"We assessed the number of high surveillance locations, those with 0 imported-and-reported cases, and low surveillance locations, those with case counts >1 Table . For our model, we assumed that the cumulative imported-and-reported case counts across 49 high surveillance locations follow a Poisson distribution from the beginning of the epidemic until February 4, 2020. Then the expected case count is linearly proportional to the daily air travel volume in the following formula:where i denotes location, Ci denotes the imported-and-reported case count in a location, λi denotes the expected case count in a location, β denotes the regression coefficient, and xi denotes the daily air travel volume of a location. The Poisson model assumes cases are independent and that the variance is equal to the expected case count. Imported-and-reported cases likely meet the independence assumption because the value excludes cases with local transmission. We also checked the robustness of our results by using an over dispersed model with a negative binomial likelihood.",
"Imported-and-reported cases likely meet the independence assumption because the value excludes cases with local transmission. We also checked the robustness of our results by using an over dispersed model with a negative binomial likelihood. We computed the p value of the overdispersion parameter as shown in Gelman and Hill .. Thumbnail of Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in number of persons/day. No. cases refers to possible undetected imported SARS-CoV-2 cases. Solid line indicates the expected imported-and-reported case counts for locations. Dashed lines represent 95% prediction interval bounds smoothed for all locations.",
"Solid line indicates the expected imported-and-reported case counts for locations. Dashed lines represent 95% prediction interval bounds smoothed for all locations. Purple dots indicate location Figure 1. Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in number of... We used R version 3.6.1 Link to compute , the maximum likelihood estimate of β, and the expected imported-and-reported case count given high surveillance Figure 1 . We also computed the 95% prediction interval PI bounds under this model of high surveillance for all 194 values of daily air travel volume Figure 1 .",
"Air travel volume measured in number of... We used R version 3.6.1 Link to compute , the maximum likelihood estimate of β, and the expected imported-and-reported case count given high surveillance Figure 1 . We also computed the 95% prediction interval PI bounds under this model of high surveillance for all 194 values of daily air travel volume Figure 1 . First, we generated a bootstrapped dataset by sampling n locations with replacement among high surveillance locations. Then, we reestimated β by using the bootstrapped dataset. Finally, we simulated imported-and-reported case counts for all 194 locations under our model by using the estimate of β from the bootstrapped dataset. We repeated the 3 steps 50,000 times to generate 50,000 simulated imported-and-reported case counts for each of the locations computed to the lower and upper PI bounds PI 2.5%–97.5% .",
"Finally, we simulated imported-and-reported case counts for all 194 locations under our model by using the estimate of β from the bootstrapped dataset. We repeated the 3 steps 50,000 times to generate 50,000 simulated imported-and-reported case counts for each of the locations computed to the lower and upper PI bounds PI 2.5%–97.5% . We smoothed the 95% PI bounds by using ggplot2 in R .. We fit the imported-and-reported case counts of the 49 high surveillance locations to the model and plotted these alongside 145 locations with low surveillance capacity Figure 1 . We noted some overlap between high and low surveillance locations Figure 1 . Thumbnail of Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in number of persons/day.",
"Thumbnail of Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in number of persons/day. No. cases refers to possible undetected imported SARS-CoV-2 cases. Solid line shows the expected imported-and-reported case counts based on our model fitted to high surveillance locations, indicated by purple dots. Dashed lines indicate the 95% pr Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 .",
"Dashed lines indicate the 95% pr Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in... To assess the robustness of our results we ran 8 additional regression analyses by implementing a series of changes to the analysis. The changes included the following: set the daily air travel volume to 0.1, 1, or 6 passengers/day for locations not listed by Lai et al. unpub. data, Link Figure 2, panels A–C ; removed all locations not listed by Lai et al.",
"unpub. data, Link Figure 2, panels A–C ; removed all locations not listed by Lai et al. before fitting Figure 2, panel D ; defined high surveillance locations by using a more lenient GHS Index criterion, 50th quantile Figure 2, panel E , and a more stringent criterion, 95th quantile Figure 2, panel F ; excluded Thailand from the model because it is a high-leverage point Figure 2, panel G ; or used an overdispersed Poisson likelihood with a negative-binomial likelihood Figure 2, panel H . We provide code for these analyses on GitHub Link . Top Results We found that daily air travel volume positively correlates with imported-and-reported case counts of SARS-CoV-2 infection among high surveillance locations Figure 1 . We noted that increasing flight volume by 31 passengers/day is associated with 1 additional expected imported-and-reported case.",
"Top Results We found that daily air travel volume positively correlates with imported-and-reported case counts of SARS-CoV-2 infection among high surveillance locations Figure 1 . We noted that increasing flight volume by 31 passengers/day is associated with 1 additional expected imported-and-reported case. In addition, Singapore and India lie above the 95% PI in our model; Singapore had 12 more imported-and-reported cases 95% PI 6–17 cases than expected and India had 3 95% PI 1–3 cases more than expected. Thailand has a relatively high air travel volume compared with other locations, but it lies below the 95% PI, reporting 16 95% PI 1–40 cases fewer imported-and-reported cases than expected under the model. Indonesia lies below the PI and has no imported-and-reported cases, but the expected case count is 5 95% PI 1–10 cases in our model. Across all 8 robustness regression analyses, we consistently observed that Singapore lies above the 95% PI and Thailand and Indonesia lie below Figure 2 .",
"Indonesia lies below the PI and has no imported-and-reported cases, but the expected case count is 5 95% PI 1–10 cases in our model. Across all 8 robustness regression analyses, we consistently observed that Singapore lies above the 95% PI and Thailand and Indonesia lie below Figure 2 . India remains above the 95% PI in all robustness analyses except when we used the more stringent GHS Index, 95th quantile, for fitting; then India lies on the upper bound of the 95% PI Figure 2, panel F . Top Discussion We aimed to identify locations with likely undetected or underdetected imported cases of SARS-CoV-2 by fitting a model to the case counts in locations with high surveillance capacity and Wuhan-to-location air travel volumes. Our model can be adjusted to account for exportation of cases from locations other than Wuhan as the outbreak develops and more information on importations and self-sustained transmission becomes available. One key advantage of this model is that it does not rely on estimates of incidence or prevalence in the epicenter of the outbreak.",
"Our model can be adjusted to account for exportation of cases from locations other than Wuhan as the outbreak develops and more information on importations and self-sustained transmission becomes available. One key advantage of this model is that it does not rely on estimates of incidence or prevalence in the epicenter of the outbreak. Also, we intentionally used a simple generalized linear model. The linearity of the expected case count means that we have only 1 regression coefficient in the model and no extra parameters. The Poisson likelihood then captures the many 0-counts observed for less highly connected locations but also describes the slope between case-count and flight data among more connected locations. We believe this model provides the most parsimonious phenomenologic description of the data.",
"The Poisson likelihood then captures the many 0-counts observed for less highly connected locations but also describes the slope between case-count and flight data among more connected locations. We believe this model provides the most parsimonious phenomenologic description of the data. According to our model, locations above the 95% PI of imported-and-reported cases could have higher case-detection capacity. Locations below the 95% PI might have undetected cases because of expected imported-and-reported case counts under high surveillance. Underdetection of cases could increase the international spread of the outbreak because the transmission chain could be lost, reducing opportunities to deploy case-based control strategies. We recommend rapid strengthening of outbreak surveillance and control efforts in locations below the 95% PI lower bound, particularly Indonesia, to curb potential local transmission.",
"Underdetection of cases could increase the international spread of the outbreak because the transmission chain could be lost, reducing opportunities to deploy case-based control strategies. We recommend rapid strengthening of outbreak surveillance and control efforts in locations below the 95% PI lower bound, particularly Indonesia, to curb potential local transmission. Early detection of cases and implantation of appropriate control measures can reduce the risk for self-sustained transmission in all locations. Top Dr. De Salazar is a research fellow at Harvard T.H. Chan School of Public Health, working on multiscale statistical models of infectious diseases within host, population, and metapopulation models. His research interests include diagnostic laboratory methods and public health response.",
"Chan School of Public Health, working on multiscale statistical models of infectious diseases within host, population, and metapopulation models. His research interests include diagnostic laboratory methods and public health response. Top Acknowledgments We thank Pamela Martinez, Nicholas Jewel, and Stephen Kissler for valuable feedback. This work was supported by US National Institute of General Medical Sciences award no. U54GM088558 . P.M.D was supported by the Fellowship Foundation Ramon Areces. A.R.T. and C.O.B. were supported by a Maximizing Investigator’s Research Award no. R35GM124715-02 from the US National Institute of General Medical Sciences.",
"A.R.T. and C.O.B. were supported by a Maximizing Investigator’s Research Award no. R35GM124715-02 from the US National Institute of General Medical Sciences. The authors are solely responsible for this content and it does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. Declaration of interests: Marc Lipsitch has received consulting fees from Merck. All other authors declare no competing interests. Top References Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–3.",
"A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–3. Wu JT, Leung K, Leung GM. Nowcasting and forecasting the potential domestic and international spread of the 2019-nCoV outbreak originating in Wuhan, China: a modelling study. Lancet. 2020;395:689–97. DOIExternal LinkPubMedExternal Link World Health Organization. Coronavirus disease 2019 COVID-19 situation report—15, 4 Feb 2020 cited 2020 Feb 14 . Link Nuclear Threat Initiative and Johns Hopkins Center for Health Security. Global health security index cited 2020 Feb 14 . Link US Environmental Protection Agency. Data quality assessment: statistical methods for practitioners EPA QA/G9-S cited 2020 Feb 14 .",
"Global health security index cited 2020 Feb 14 . Link US Environmental Protection Agency. Data quality assessment: statistical methods for practitioners EPA QA/G9-S cited 2020 Feb 14 . Washington: The Agency; 2006. Link Lamichhane S, Sen P, Dickens AM, Hyötyläinen T, Orešič M. An overview of metabolomics data analysis: current tools and future perspectives. In: Jaumot J, Bedia C, Tauler R, editors. Comprehensive analytical chemistry. Vol. 82. Amsterdam: Elsevier; 2018. p. 387–413. Gelman A, Hill J. Analytical methods for social research. In: Data analysis using regression and multilevel/hierarchical models. Cambridge: Cambridge University Press; 2006. p. 235–236.",
"Gelman A, Hill J. Analytical methods for social research. In: Data analysis using regression and multilevel/hierarchical models. Cambridge: Cambridge University Press; 2006. p. 235–236. Wickham H. ggplot2: elegant graphics for data analysis. New York: Springer; 2016. Top Figures Figure 1. Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in... Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume... Table Table.",
"Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume... Table Table. Surveillance capacity of locations with and without imported-and-reported cases of severe acute respiratory syndrome coronavirus 2, 2020 Top Suggested citation for this article: De Salazar PM, Niehus R, Taylor A, O’Flaherty Buckee C, Lipsitch M. Identifying locations with possible undetected imported severe acute respiratory syndrome coronavirus 2 cases by using importation predictions. Emerg Infect Dis. 2020 Jul date cited . DOI: 10.3201/eid2607.200250 Original Publication Date: 3/24/2020 1These authors contributed equally to this article.",
"Emerg Infect Dis. 2020 Jul date cited . DOI: 10.3201/eid2607.200250 Original Publication Date: 3/24/2020 1These authors contributed equally to this article. Table of Contents – Volume 26, Number 7—July 2020"
] | 186 | 244 |
When was SARS-CoV-2 first identified? | December 2019 | [
"Identifying Locations with Possible Undetected Imported Severe Acute Respiratory Syndrome Coronavirus 2 Cases by Using Importation Predictions, Volume 26, Number 7—July 2020 Research Pablo Martinez De Salazar1Comments to Author , René Niehus, Aimee Taylor1, Caroline O’Flaherty Buckee, and Marc LipsitchComments to Author Author affiliations: Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA Suggested citation for this article Abstract Cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 infection exported from mainland China could lead to self-sustained outbreaks in other countries. By February 2020, several countries were reporting imported SARS-CoV-2 cases. To contain the virus, early detection of imported SARS-CoV-2 cases is critical. We used air travel volume estimates from Wuhan, China, to international destinations and a generalized linear regression model to identify locations that could have undetected imported cases. Our model can be adjusted to account for exportation of cases from other locations as the virus spreads and more information on importations and transmission becomes available.",
"We used air travel volume estimates from Wuhan, China, to international destinations and a generalized linear regression model to identify locations that could have undetected imported cases. Our model can be adjusted to account for exportation of cases from other locations as the virus spreads and more information on importations and transmission becomes available. Early detection and appropriate control measures can reduce the risk for transmission in all locations. A novel coronavirus, later named severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 , was identified in December 2019 in the city of Wuhan, capital of Hubei Province, China, where cases were first confirmed .. During December 2019–February 2020, the number of confirmed cases increased drastically. Model estimates suggested that >75,000 persons were infected by January 25, 2020, and the epidemic had a doubling time of ≈6 days .. By the end of January 2020, travel restrictions were implemented for Wuhan and neighboring cities. Nonetheless, the virus spread from Wuhan to other cities in China and outside the country.",
"Model estimates suggested that >75,000 persons were infected by January 25, 2020, and the epidemic had a doubling time of ≈6 days .. By the end of January 2020, travel restrictions were implemented for Wuhan and neighboring cities. Nonetheless, the virus spread from Wuhan to other cities in China and outside the country. By February 4, 2020, a total of 23 locations outside mainland China reported cases, 22 of which reported imported cases; Spain reported a case caused by secondary transmission .. Most cases imported to other locations have been linked to recent travel history from China ., suggesting that air travel plays a major role in exportation of cases to locations outside of China. To prevent other cities and countries from becoming epicenters of the SARS-CoV-2 epidemic, substantial targeted public health interventions are required to detect cases and control local spread of the virus. We collected estimates of air travel volume from Wuhan to 194 international destinations.",
"To prevent other cities and countries from becoming epicenters of the SARS-CoV-2 epidemic, substantial targeted public health interventions are required to detect cases and control local spread of the virus. We collected estimates of air travel volume from Wuhan to 194 international destinations. We then identified 49 countries that had a score of >49.2/100 on category 2, Early Detection and Reporting of Epidemics of Potential International Concern, of the Global Health Security GHS Index .. We assumed these locations would be proficient at detecting SARS-CoV-2 and reporting confirmed imported cases, which we refer to as imported-and-reported cases. We ran a generalized linear regression model on this subset; based on the results, we generated predictions for the remainder of the sample. Using these predictions, we identified locations that might not be detecting imported cases. Methods To identify locations reporting fewer than predicted imported SARS-CoV-2 infected cases, we fit a model to data from 49 locations outside mainland China with high surveillance capacity according to the GHS Index ..",
"Using these predictions, we identified locations that might not be detecting imported cases. Methods To identify locations reporting fewer than predicted imported SARS-CoV-2 infected cases, we fit a model to data from 49 locations outside mainland China with high surveillance capacity according to the GHS Index .. Among these, 17 had high travel connectivity to Wuhan and 32 have low connectivity to Wuhan. We considered locations to be countries without any position on territorial claims. We performed a Poisson regression by using the cumulative number of imported-and-reported SARS-CoV-2 cases in these 49 countries and the estimated number of daily airline passengers from the Wuhan airport. We then compared predictions from this model with imported-and-reported cases across 194 locations from the GHS Index, excluding China as the epicenter of the outbreak.",
"We performed a Poisson regression by using the cumulative number of imported-and-reported SARS-CoV-2 cases in these 49 countries and the estimated number of daily airline passengers from the Wuhan airport. We then compared predictions from this model with imported-and-reported cases across 194 locations from the GHS Index, excluding China as the epicenter of the outbreak. The model requires data on imported-and-reported cases of SARS-CoV-2 infection, daily air travel volume, and surveillance capacity. We obtained data on imported-and-reported cases aggregated by destination from the World Health Organization technical report issued February 4, 2020 .. We assumed a case count of 0 for locations not listed. We used February 4 as the cutoff for cumulative imported-and-reported case counts because exported cases from Hubei Province dropped rapidly after this date ., likely because of travel restrictions for the province implement on January 23. We defined imported-and-reported cases as those with known travel history from China; of those, 83% had a travel history from Hubei Province and 17% traveled from unknown locations in China .. We excluded reported cases likely caused by transmission outside of China or cases in which the transmission source was still under investigation ..",
"We used February 4 as the cutoff for cumulative imported-and-reported case counts because exported cases from Hubei Province dropped rapidly after this date ., likely because of travel restrictions for the province implement on January 23. We defined imported-and-reported cases as those with known travel history from China; of those, 83% had a travel history from Hubei Province and 17% traveled from unknown locations in China .. We excluded reported cases likely caused by transmission outside of China or cases in which the transmission source was still under investigation .. In addition, we excluded Hong Kong, Macau, and Taiwan from our model because locally transmitted and imported cases were not disaggregated in these locations. We obtained data on daily air travel from a network-based modeling study S. Lai et al., unpub. data, Link that reported monthly air travel volume estimates for the 27 locations outside mainland China that are most connected to Wuhan. These estimates were calculated from International Air Travel Association data from February 2018, which includes direct and indirect flight itineraries from Wuhan.",
"data, Link that reported monthly air travel volume estimates for the 27 locations outside mainland China that are most connected to Wuhan. These estimates were calculated from International Air Travel Association data from February 2018, which includes direct and indirect flight itineraries from Wuhan. For these 27 locations, estimated air travel volumes are >6 passengers/day. We assumed that travel volumes for locations not among the most connected are censored by a detection limit. We used a common method of dealing with censored data from environmental sampling ., or metabolomics ., to set the daily air travel volume to half the minimum previously reported. Therefore, we used 3 passengers/day for estimated travel volumes for the 167 locations from the GHS Index not listed by Lai et al.",
"We used a common method of dealing with censored data from environmental sampling ., or metabolomics ., to set the daily air travel volume to half the minimum previously reported. Therefore, we used 3 passengers/day for estimated travel volumes for the 167 locations from the GHS Index not listed by Lai et al. We tested the robustness of our results by using a set of alternative values of 0.1, 1, and 6 passengers/day for the censored data. We defined high surveillance locations as those with a GHS Index for category 2 above the 75th quantile. We assessed the number of high surveillance locations, those with 0 imported-and-reported cases, and low surveillance locations, those with case counts >1 Table . For our model, we assumed that the cumulative imported-and-reported case counts across 49 high surveillance locations follow a Poisson distribution from the beginning of the epidemic until February 4, 2020.",
"We assessed the number of high surveillance locations, those with 0 imported-and-reported cases, and low surveillance locations, those with case counts >1 Table . For our model, we assumed that the cumulative imported-and-reported case counts across 49 high surveillance locations follow a Poisson distribution from the beginning of the epidemic until February 4, 2020. Then the expected case count is linearly proportional to the daily air travel volume in the following formula:where i denotes location, Ci denotes the imported-and-reported case count in a location, λi denotes the expected case count in a location, β denotes the regression coefficient, and xi denotes the daily air travel volume of a location. The Poisson model assumes cases are independent and that the variance is equal to the expected case count. Imported-and-reported cases likely meet the independence assumption because the value excludes cases with local transmission. We also checked the robustness of our results by using an over dispersed model with a negative binomial likelihood.",
"Imported-and-reported cases likely meet the independence assumption because the value excludes cases with local transmission. We also checked the robustness of our results by using an over dispersed model with a negative binomial likelihood. We computed the p value of the overdispersion parameter as shown in Gelman and Hill .. Thumbnail of Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in number of persons/day. No. cases refers to possible undetected imported SARS-CoV-2 cases. Solid line indicates the expected imported-and-reported case counts for locations. Dashed lines represent 95% prediction interval bounds smoothed for all locations.",
"Solid line indicates the expected imported-and-reported case counts for locations. Dashed lines represent 95% prediction interval bounds smoothed for all locations. Purple dots indicate location Figure 1. Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in number of... We used R version 3.6.1 Link to compute , the maximum likelihood estimate of β, and the expected imported-and-reported case count given high surveillance Figure 1 . We also computed the 95% prediction interval PI bounds under this model of high surveillance for all 194 values of daily air travel volume Figure 1 .",
"Air travel volume measured in number of... We used R version 3.6.1 Link to compute , the maximum likelihood estimate of β, and the expected imported-and-reported case count given high surveillance Figure 1 . We also computed the 95% prediction interval PI bounds under this model of high surveillance for all 194 values of daily air travel volume Figure 1 . First, we generated a bootstrapped dataset by sampling n locations with replacement among high surveillance locations. Then, we reestimated β by using the bootstrapped dataset. Finally, we simulated imported-and-reported case counts for all 194 locations under our model by using the estimate of β from the bootstrapped dataset. We repeated the 3 steps 50,000 times to generate 50,000 simulated imported-and-reported case counts for each of the locations computed to the lower and upper PI bounds PI 2.5%–97.5% .",
"Finally, we simulated imported-and-reported case counts for all 194 locations under our model by using the estimate of β from the bootstrapped dataset. We repeated the 3 steps 50,000 times to generate 50,000 simulated imported-and-reported case counts for each of the locations computed to the lower and upper PI bounds PI 2.5%–97.5% . We smoothed the 95% PI bounds by using ggplot2 in R .. We fit the imported-and-reported case counts of the 49 high surveillance locations to the model and plotted these alongside 145 locations with low surveillance capacity Figure 1 . We noted some overlap between high and low surveillance locations Figure 1 . Thumbnail of Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in number of persons/day.",
"Thumbnail of Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in number of persons/day. No. cases refers to possible undetected imported SARS-CoV-2 cases. Solid line shows the expected imported-and-reported case counts based on our model fitted to high surveillance locations, indicated by purple dots. Dashed lines indicate the 95% pr Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 .",
"Dashed lines indicate the 95% pr Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in... To assess the robustness of our results we ran 8 additional regression analyses by implementing a series of changes to the analysis. The changes included the following: set the daily air travel volume to 0.1, 1, or 6 passengers/day for locations not listed by Lai et al. unpub. data, Link Figure 2, panels A–C ; removed all locations not listed by Lai et al.",
"unpub. data, Link Figure 2, panels A–C ; removed all locations not listed by Lai et al. before fitting Figure 2, panel D ; defined high surveillance locations by using a more lenient GHS Index criterion, 50th quantile Figure 2, panel E , and a more stringent criterion, 95th quantile Figure 2, panel F ; excluded Thailand from the model because it is a high-leverage point Figure 2, panel G ; or used an overdispersed Poisson likelihood with a negative-binomial likelihood Figure 2, panel H . We provide code for these analyses on GitHub Link . Top Results We found that daily air travel volume positively correlates with imported-and-reported case counts of SARS-CoV-2 infection among high surveillance locations Figure 1 . We noted that increasing flight volume by 31 passengers/day is associated with 1 additional expected imported-and-reported case.",
"Top Results We found that daily air travel volume positively correlates with imported-and-reported case counts of SARS-CoV-2 infection among high surveillance locations Figure 1 . We noted that increasing flight volume by 31 passengers/day is associated with 1 additional expected imported-and-reported case. In addition, Singapore and India lie above the 95% PI in our model; Singapore had 12 more imported-and-reported cases 95% PI 6–17 cases than expected and India had 3 95% PI 1–3 cases more than expected. Thailand has a relatively high air travel volume compared with other locations, but it lies below the 95% PI, reporting 16 95% PI 1–40 cases fewer imported-and-reported cases than expected under the model. Indonesia lies below the PI and has no imported-and-reported cases, but the expected case count is 5 95% PI 1–10 cases in our model. Across all 8 robustness regression analyses, we consistently observed that Singapore lies above the 95% PI and Thailand and Indonesia lie below Figure 2 .",
"Indonesia lies below the PI and has no imported-and-reported cases, but the expected case count is 5 95% PI 1–10 cases in our model. Across all 8 robustness regression analyses, we consistently observed that Singapore lies above the 95% PI and Thailand and Indonesia lie below Figure 2 . India remains above the 95% PI in all robustness analyses except when we used the more stringent GHS Index, 95th quantile, for fitting; then India lies on the upper bound of the 95% PI Figure 2, panel F . Top Discussion We aimed to identify locations with likely undetected or underdetected imported cases of SARS-CoV-2 by fitting a model to the case counts in locations with high surveillance capacity and Wuhan-to-location air travel volumes. Our model can be adjusted to account for exportation of cases from locations other than Wuhan as the outbreak develops and more information on importations and self-sustained transmission becomes available. One key advantage of this model is that it does not rely on estimates of incidence or prevalence in the epicenter of the outbreak.",
"Our model can be adjusted to account for exportation of cases from locations other than Wuhan as the outbreak develops and more information on importations and self-sustained transmission becomes available. One key advantage of this model is that it does not rely on estimates of incidence or prevalence in the epicenter of the outbreak. Also, we intentionally used a simple generalized linear model. The linearity of the expected case count means that we have only 1 regression coefficient in the model and no extra parameters. The Poisson likelihood then captures the many 0-counts observed for less highly connected locations but also describes the slope between case-count and flight data among more connected locations. We believe this model provides the most parsimonious phenomenologic description of the data.",
"The Poisson likelihood then captures the many 0-counts observed for less highly connected locations but also describes the slope between case-count and flight data among more connected locations. We believe this model provides the most parsimonious phenomenologic description of the data. According to our model, locations above the 95% PI of imported-and-reported cases could have higher case-detection capacity. Locations below the 95% PI might have undetected cases because of expected imported-and-reported case counts under high surveillance. Underdetection of cases could increase the international spread of the outbreak because the transmission chain could be lost, reducing opportunities to deploy case-based control strategies. We recommend rapid strengthening of outbreak surveillance and control efforts in locations below the 95% PI lower bound, particularly Indonesia, to curb potential local transmission.",
"Underdetection of cases could increase the international spread of the outbreak because the transmission chain could be lost, reducing opportunities to deploy case-based control strategies. We recommend rapid strengthening of outbreak surveillance and control efforts in locations below the 95% PI lower bound, particularly Indonesia, to curb potential local transmission. Early detection of cases and implantation of appropriate control measures can reduce the risk for self-sustained transmission in all locations. Top Dr. De Salazar is a research fellow at Harvard T.H. Chan School of Public Health, working on multiscale statistical models of infectious diseases within host, population, and metapopulation models. His research interests include diagnostic laboratory methods and public health response.",
"Chan School of Public Health, working on multiscale statistical models of infectious diseases within host, population, and metapopulation models. His research interests include diagnostic laboratory methods and public health response. Top Acknowledgments We thank Pamela Martinez, Nicholas Jewel, and Stephen Kissler for valuable feedback. This work was supported by US National Institute of General Medical Sciences award no. U54GM088558 . P.M.D was supported by the Fellowship Foundation Ramon Areces. A.R.T. and C.O.B. were supported by a Maximizing Investigator’s Research Award no. R35GM124715-02 from the US National Institute of General Medical Sciences.",
"A.R.T. and C.O.B. were supported by a Maximizing Investigator’s Research Award no. R35GM124715-02 from the US National Institute of General Medical Sciences. The authors are solely responsible for this content and it does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. Declaration of interests: Marc Lipsitch has received consulting fees from Merck. All other authors declare no competing interests. Top References Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–3.",
"A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–3. Wu JT, Leung K, Leung GM. Nowcasting and forecasting the potential domestic and international spread of the 2019-nCoV outbreak originating in Wuhan, China: a modelling study. Lancet. 2020;395:689–97. DOIExternal LinkPubMedExternal Link World Health Organization. Coronavirus disease 2019 COVID-19 situation report—15, 4 Feb 2020 cited 2020 Feb 14 . Link Nuclear Threat Initiative and Johns Hopkins Center for Health Security. Global health security index cited 2020 Feb 14 . Link US Environmental Protection Agency. Data quality assessment: statistical methods for practitioners EPA QA/G9-S cited 2020 Feb 14 .",
"Global health security index cited 2020 Feb 14 . Link US Environmental Protection Agency. Data quality assessment: statistical methods for practitioners EPA QA/G9-S cited 2020 Feb 14 . Washington: The Agency; 2006. Link Lamichhane S, Sen P, Dickens AM, Hyötyläinen T, Orešič M. An overview of metabolomics data analysis: current tools and future perspectives. In: Jaumot J, Bedia C, Tauler R, editors. Comprehensive analytical chemistry. Vol. 82. Amsterdam: Elsevier; 2018. p. 387–413. Gelman A, Hill J. Analytical methods for social research. In: Data analysis using regression and multilevel/hierarchical models. Cambridge: Cambridge University Press; 2006. p. 235–236.",
"Gelman A, Hill J. Analytical methods for social research. In: Data analysis using regression and multilevel/hierarchical models. Cambridge: Cambridge University Press; 2006. p. 235–236. Wickham H. ggplot2: elegant graphics for data analysis. New York: Springer; 2016. Top Figures Figure 1. Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in... Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume... Table Table.",
"Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume... Table Table. Surveillance capacity of locations with and without imported-and-reported cases of severe acute respiratory syndrome coronavirus 2, 2020 Top Suggested citation for this article: De Salazar PM, Niehus R, Taylor A, O’Flaherty Buckee C, Lipsitch M. Identifying locations with possible undetected imported severe acute respiratory syndrome coronavirus 2 cases by using importation predictions. Emerg Infect Dis. 2020 Jul date cited . DOI: 10.3201/eid2607.200250 Original Publication Date: 3/24/2020 1These authors contributed equally to this article.",
"Emerg Infect Dis. 2020 Jul date cited . DOI: 10.3201/eid2607.200250 Original Publication Date: 3/24/2020 1These authors contributed equally to this article. Table of Contents – Volume 26, Number 7—July 2020"
] | 186 | 245 |
Where was SARS-CoV-2 first identified? | Wuhan, capital of Hubei Province, China | [
"Identifying Locations with Possible Undetected Imported Severe Acute Respiratory Syndrome Coronavirus 2 Cases by Using Importation Predictions, Volume 26, Number 7—July 2020 Research Pablo Martinez De Salazar1Comments to Author , René Niehus, Aimee Taylor1, Caroline O’Flaherty Buckee, and Marc LipsitchComments to Author Author affiliations: Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA Suggested citation for this article Abstract Cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 infection exported from mainland China could lead to self-sustained outbreaks in other countries. By February 2020, several countries were reporting imported SARS-CoV-2 cases. To contain the virus, early detection of imported SARS-CoV-2 cases is critical. We used air travel volume estimates from Wuhan, China, to international destinations and a generalized linear regression model to identify locations that could have undetected imported cases. Our model can be adjusted to account for exportation of cases from other locations as the virus spreads and more information on importations and transmission becomes available.",
"We used air travel volume estimates from Wuhan, China, to international destinations and a generalized linear regression model to identify locations that could have undetected imported cases. Our model can be adjusted to account for exportation of cases from other locations as the virus spreads and more information on importations and transmission becomes available. Early detection and appropriate control measures can reduce the risk for transmission in all locations. A novel coronavirus, later named severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 , was identified in December 2019 in the city of Wuhan, capital of Hubei Province, China, where cases were first confirmed .. During December 2019–February 2020, the number of confirmed cases increased drastically. Model estimates suggested that >75,000 persons were infected by January 25, 2020, and the epidemic had a doubling time of ≈6 days .. By the end of January 2020, travel restrictions were implemented for Wuhan and neighboring cities. Nonetheless, the virus spread from Wuhan to other cities in China and outside the country.",
"Model estimates suggested that >75,000 persons were infected by January 25, 2020, and the epidemic had a doubling time of ≈6 days .. By the end of January 2020, travel restrictions were implemented for Wuhan and neighboring cities. Nonetheless, the virus spread from Wuhan to other cities in China and outside the country. By February 4, 2020, a total of 23 locations outside mainland China reported cases, 22 of which reported imported cases; Spain reported a case caused by secondary transmission .. Most cases imported to other locations have been linked to recent travel history from China ., suggesting that air travel plays a major role in exportation of cases to locations outside of China. To prevent other cities and countries from becoming epicenters of the SARS-CoV-2 epidemic, substantial targeted public health interventions are required to detect cases and control local spread of the virus. We collected estimates of air travel volume from Wuhan to 194 international destinations.",
"To prevent other cities and countries from becoming epicenters of the SARS-CoV-2 epidemic, substantial targeted public health interventions are required to detect cases and control local spread of the virus. We collected estimates of air travel volume from Wuhan to 194 international destinations. We then identified 49 countries that had a score of >49.2/100 on category 2, Early Detection and Reporting of Epidemics of Potential International Concern, of the Global Health Security GHS Index .. We assumed these locations would be proficient at detecting SARS-CoV-2 and reporting confirmed imported cases, which we refer to as imported-and-reported cases. We ran a generalized linear regression model on this subset; based on the results, we generated predictions for the remainder of the sample. Using these predictions, we identified locations that might not be detecting imported cases. Methods To identify locations reporting fewer than predicted imported SARS-CoV-2 infected cases, we fit a model to data from 49 locations outside mainland China with high surveillance capacity according to the GHS Index ..",
"Using these predictions, we identified locations that might not be detecting imported cases. Methods To identify locations reporting fewer than predicted imported SARS-CoV-2 infected cases, we fit a model to data from 49 locations outside mainland China with high surveillance capacity according to the GHS Index .. Among these, 17 had high travel connectivity to Wuhan and 32 have low connectivity to Wuhan. We considered locations to be countries without any position on territorial claims. We performed a Poisson regression by using the cumulative number of imported-and-reported SARS-CoV-2 cases in these 49 countries and the estimated number of daily airline passengers from the Wuhan airport. We then compared predictions from this model with imported-and-reported cases across 194 locations from the GHS Index, excluding China as the epicenter of the outbreak.",
"We performed a Poisson regression by using the cumulative number of imported-and-reported SARS-CoV-2 cases in these 49 countries and the estimated number of daily airline passengers from the Wuhan airport. We then compared predictions from this model with imported-and-reported cases across 194 locations from the GHS Index, excluding China as the epicenter of the outbreak. The model requires data on imported-and-reported cases of SARS-CoV-2 infection, daily air travel volume, and surveillance capacity. We obtained data on imported-and-reported cases aggregated by destination from the World Health Organization technical report issued February 4, 2020 .. We assumed a case count of 0 for locations not listed. We used February 4 as the cutoff for cumulative imported-and-reported case counts because exported cases from Hubei Province dropped rapidly after this date ., likely because of travel restrictions for the province implement on January 23. We defined imported-and-reported cases as those with known travel history from China; of those, 83% had a travel history from Hubei Province and 17% traveled from unknown locations in China .. We excluded reported cases likely caused by transmission outside of China or cases in which the transmission source was still under investigation ..",
"We used February 4 as the cutoff for cumulative imported-and-reported case counts because exported cases from Hubei Province dropped rapidly after this date ., likely because of travel restrictions for the province implement on January 23. We defined imported-and-reported cases as those with known travel history from China; of those, 83% had a travel history from Hubei Province and 17% traveled from unknown locations in China .. We excluded reported cases likely caused by transmission outside of China or cases in which the transmission source was still under investigation .. In addition, we excluded Hong Kong, Macau, and Taiwan from our model because locally transmitted and imported cases were not disaggregated in these locations. We obtained data on daily air travel from a network-based modeling study S. Lai et al., unpub. data, Link that reported monthly air travel volume estimates for the 27 locations outside mainland China that are most connected to Wuhan. These estimates were calculated from International Air Travel Association data from February 2018, which includes direct and indirect flight itineraries from Wuhan.",
"data, Link that reported monthly air travel volume estimates for the 27 locations outside mainland China that are most connected to Wuhan. These estimates were calculated from International Air Travel Association data from February 2018, which includes direct and indirect flight itineraries from Wuhan. For these 27 locations, estimated air travel volumes are >6 passengers/day. We assumed that travel volumes for locations not among the most connected are censored by a detection limit. We used a common method of dealing with censored data from environmental sampling ., or metabolomics ., to set the daily air travel volume to half the minimum previously reported. Therefore, we used 3 passengers/day for estimated travel volumes for the 167 locations from the GHS Index not listed by Lai et al.",
"We used a common method of dealing with censored data from environmental sampling ., or metabolomics ., to set the daily air travel volume to half the minimum previously reported. Therefore, we used 3 passengers/day for estimated travel volumes for the 167 locations from the GHS Index not listed by Lai et al. We tested the robustness of our results by using a set of alternative values of 0.1, 1, and 6 passengers/day for the censored data. We defined high surveillance locations as those with a GHS Index for category 2 above the 75th quantile. We assessed the number of high surveillance locations, those with 0 imported-and-reported cases, and low surveillance locations, those with case counts >1 Table . For our model, we assumed that the cumulative imported-and-reported case counts across 49 high surveillance locations follow a Poisson distribution from the beginning of the epidemic until February 4, 2020.",
"We assessed the number of high surveillance locations, those with 0 imported-and-reported cases, and low surveillance locations, those with case counts >1 Table . For our model, we assumed that the cumulative imported-and-reported case counts across 49 high surveillance locations follow a Poisson distribution from the beginning of the epidemic until February 4, 2020. Then the expected case count is linearly proportional to the daily air travel volume in the following formula:where i denotes location, Ci denotes the imported-and-reported case count in a location, λi denotes the expected case count in a location, β denotes the regression coefficient, and xi denotes the daily air travel volume of a location. The Poisson model assumes cases are independent and that the variance is equal to the expected case count. Imported-and-reported cases likely meet the independence assumption because the value excludes cases with local transmission. We also checked the robustness of our results by using an over dispersed model with a negative binomial likelihood.",
"Imported-and-reported cases likely meet the independence assumption because the value excludes cases with local transmission. We also checked the robustness of our results by using an over dispersed model with a negative binomial likelihood. We computed the p value of the overdispersion parameter as shown in Gelman and Hill .. Thumbnail of Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in number of persons/day. No. cases refers to possible undetected imported SARS-CoV-2 cases. Solid line indicates the expected imported-and-reported case counts for locations. Dashed lines represent 95% prediction interval bounds smoothed for all locations.",
"Solid line indicates the expected imported-and-reported case counts for locations. Dashed lines represent 95% prediction interval bounds smoothed for all locations. Purple dots indicate location Figure 1. Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in number of... We used R version 3.6.1 Link to compute , the maximum likelihood estimate of β, and the expected imported-and-reported case count given high surveillance Figure 1 . We also computed the 95% prediction interval PI bounds under this model of high surveillance for all 194 values of daily air travel volume Figure 1 .",
"Air travel volume measured in number of... We used R version 3.6.1 Link to compute , the maximum likelihood estimate of β, and the expected imported-and-reported case count given high surveillance Figure 1 . We also computed the 95% prediction interval PI bounds under this model of high surveillance for all 194 values of daily air travel volume Figure 1 . First, we generated a bootstrapped dataset by sampling n locations with replacement among high surveillance locations. Then, we reestimated β by using the bootstrapped dataset. Finally, we simulated imported-and-reported case counts for all 194 locations under our model by using the estimate of β from the bootstrapped dataset. We repeated the 3 steps 50,000 times to generate 50,000 simulated imported-and-reported case counts for each of the locations computed to the lower and upper PI bounds PI 2.5%–97.5% .",
"Finally, we simulated imported-and-reported case counts for all 194 locations under our model by using the estimate of β from the bootstrapped dataset. We repeated the 3 steps 50,000 times to generate 50,000 simulated imported-and-reported case counts for each of the locations computed to the lower and upper PI bounds PI 2.5%–97.5% . We smoothed the 95% PI bounds by using ggplot2 in R .. We fit the imported-and-reported case counts of the 49 high surveillance locations to the model and plotted these alongside 145 locations with low surveillance capacity Figure 1 . We noted some overlap between high and low surveillance locations Figure 1 . Thumbnail of Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in number of persons/day.",
"Thumbnail of Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in number of persons/day. No. cases refers to possible undetected imported SARS-CoV-2 cases. Solid line shows the expected imported-and-reported case counts based on our model fitted to high surveillance locations, indicated by purple dots. Dashed lines indicate the 95% pr Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 .",
"Dashed lines indicate the 95% pr Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in... To assess the robustness of our results we ran 8 additional regression analyses by implementing a series of changes to the analysis. The changes included the following: set the daily air travel volume to 0.1, 1, or 6 passengers/day for locations not listed by Lai et al. unpub. data, Link Figure 2, panels A–C ; removed all locations not listed by Lai et al.",
"unpub. data, Link Figure 2, panels A–C ; removed all locations not listed by Lai et al. before fitting Figure 2, panel D ; defined high surveillance locations by using a more lenient GHS Index criterion, 50th quantile Figure 2, panel E , and a more stringent criterion, 95th quantile Figure 2, panel F ; excluded Thailand from the model because it is a high-leverage point Figure 2, panel G ; or used an overdispersed Poisson likelihood with a negative-binomial likelihood Figure 2, panel H . We provide code for these analyses on GitHub Link . Top Results We found that daily air travel volume positively correlates with imported-and-reported case counts of SARS-CoV-2 infection among high surveillance locations Figure 1 . We noted that increasing flight volume by 31 passengers/day is associated with 1 additional expected imported-and-reported case.",
"Top Results We found that daily air travel volume positively correlates with imported-and-reported case counts of SARS-CoV-2 infection among high surveillance locations Figure 1 . We noted that increasing flight volume by 31 passengers/day is associated with 1 additional expected imported-and-reported case. In addition, Singapore and India lie above the 95% PI in our model; Singapore had 12 more imported-and-reported cases 95% PI 6–17 cases than expected and India had 3 95% PI 1–3 cases more than expected. Thailand has a relatively high air travel volume compared with other locations, but it lies below the 95% PI, reporting 16 95% PI 1–40 cases fewer imported-and-reported cases than expected under the model. Indonesia lies below the PI and has no imported-and-reported cases, but the expected case count is 5 95% PI 1–10 cases in our model. Across all 8 robustness regression analyses, we consistently observed that Singapore lies above the 95% PI and Thailand and Indonesia lie below Figure 2 .",
"Indonesia lies below the PI and has no imported-and-reported cases, but the expected case count is 5 95% PI 1–10 cases in our model. Across all 8 robustness regression analyses, we consistently observed that Singapore lies above the 95% PI and Thailand and Indonesia lie below Figure 2 . India remains above the 95% PI in all robustness analyses except when we used the more stringent GHS Index, 95th quantile, for fitting; then India lies on the upper bound of the 95% PI Figure 2, panel F . Top Discussion We aimed to identify locations with likely undetected or underdetected imported cases of SARS-CoV-2 by fitting a model to the case counts in locations with high surveillance capacity and Wuhan-to-location air travel volumes. Our model can be adjusted to account for exportation of cases from locations other than Wuhan as the outbreak develops and more information on importations and self-sustained transmission becomes available. One key advantage of this model is that it does not rely on estimates of incidence or prevalence in the epicenter of the outbreak.",
"Our model can be adjusted to account for exportation of cases from locations other than Wuhan as the outbreak develops and more information on importations and self-sustained transmission becomes available. One key advantage of this model is that it does not rely on estimates of incidence or prevalence in the epicenter of the outbreak. Also, we intentionally used a simple generalized linear model. The linearity of the expected case count means that we have only 1 regression coefficient in the model and no extra parameters. The Poisson likelihood then captures the many 0-counts observed for less highly connected locations but also describes the slope between case-count and flight data among more connected locations. We believe this model provides the most parsimonious phenomenologic description of the data.",
"The Poisson likelihood then captures the many 0-counts observed for less highly connected locations but also describes the slope between case-count and flight data among more connected locations. We believe this model provides the most parsimonious phenomenologic description of the data. According to our model, locations above the 95% PI of imported-and-reported cases could have higher case-detection capacity. Locations below the 95% PI might have undetected cases because of expected imported-and-reported case counts under high surveillance. Underdetection of cases could increase the international spread of the outbreak because the transmission chain could be lost, reducing opportunities to deploy case-based control strategies. We recommend rapid strengthening of outbreak surveillance and control efforts in locations below the 95% PI lower bound, particularly Indonesia, to curb potential local transmission.",
"Underdetection of cases could increase the international spread of the outbreak because the transmission chain could be lost, reducing opportunities to deploy case-based control strategies. We recommend rapid strengthening of outbreak surveillance and control efforts in locations below the 95% PI lower bound, particularly Indonesia, to curb potential local transmission. Early detection of cases and implantation of appropriate control measures can reduce the risk for self-sustained transmission in all locations. Top Dr. De Salazar is a research fellow at Harvard T.H. Chan School of Public Health, working on multiscale statistical models of infectious diseases within host, population, and metapopulation models. His research interests include diagnostic laboratory methods and public health response.",
"Chan School of Public Health, working on multiscale statistical models of infectious diseases within host, population, and metapopulation models. His research interests include diagnostic laboratory methods and public health response. Top Acknowledgments We thank Pamela Martinez, Nicholas Jewel, and Stephen Kissler for valuable feedback. This work was supported by US National Institute of General Medical Sciences award no. U54GM088558 . P.M.D was supported by the Fellowship Foundation Ramon Areces. A.R.T. and C.O.B. were supported by a Maximizing Investigator’s Research Award no. R35GM124715-02 from the US National Institute of General Medical Sciences.",
"A.R.T. and C.O.B. were supported by a Maximizing Investigator’s Research Award no. R35GM124715-02 from the US National Institute of General Medical Sciences. The authors are solely responsible for this content and it does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. Declaration of interests: Marc Lipsitch has received consulting fees from Merck. All other authors declare no competing interests. Top References Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–3.",
"A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–3. Wu JT, Leung K, Leung GM. Nowcasting and forecasting the potential domestic and international spread of the 2019-nCoV outbreak originating in Wuhan, China: a modelling study. Lancet. 2020;395:689–97. DOIExternal LinkPubMedExternal Link World Health Organization. Coronavirus disease 2019 COVID-19 situation report—15, 4 Feb 2020 cited 2020 Feb 14 . Link Nuclear Threat Initiative and Johns Hopkins Center for Health Security. Global health security index cited 2020 Feb 14 . Link US Environmental Protection Agency. Data quality assessment: statistical methods for practitioners EPA QA/G9-S cited 2020 Feb 14 .",
"Global health security index cited 2020 Feb 14 . Link US Environmental Protection Agency. Data quality assessment: statistical methods for practitioners EPA QA/G9-S cited 2020 Feb 14 . Washington: The Agency; 2006. Link Lamichhane S, Sen P, Dickens AM, Hyötyläinen T, Orešič M. An overview of metabolomics data analysis: current tools and future perspectives. In: Jaumot J, Bedia C, Tauler R, editors. Comprehensive analytical chemistry. Vol. 82. Amsterdam: Elsevier; 2018. p. 387–413. Gelman A, Hill J. Analytical methods for social research. In: Data analysis using regression and multilevel/hierarchical models. Cambridge: Cambridge University Press; 2006. p. 235–236.",
"Gelman A, Hill J. Analytical methods for social research. In: Data analysis using regression and multilevel/hierarchical models. Cambridge: Cambridge University Press; 2006. p. 235–236. Wickham H. ggplot2: elegant graphics for data analysis. New York: Springer; 2016. Top Figures Figure 1. Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in... Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume... Table Table.",
"Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume... Table Table. Surveillance capacity of locations with and without imported-and-reported cases of severe acute respiratory syndrome coronavirus 2, 2020 Top Suggested citation for this article: De Salazar PM, Niehus R, Taylor A, O’Flaherty Buckee C, Lipsitch M. Identifying locations with possible undetected imported severe acute respiratory syndrome coronavirus 2 cases by using importation predictions. Emerg Infect Dis. 2020 Jul date cited . DOI: 10.3201/eid2607.200250 Original Publication Date: 3/24/2020 1These authors contributed equally to this article.",
"Emerg Infect Dis. 2020 Jul date cited . DOI: 10.3201/eid2607.200250 Original Publication Date: 3/24/2020 1These authors contributed equally to this article. Table of Contents – Volume 26, Number 7—July 2020"
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What factor positively correlates with imported-and-reported cases counts of SARS-CoV-2 infection? | daily air travel volume | [
"Identifying Locations with Possible Undetected Imported Severe Acute Respiratory Syndrome Coronavirus 2 Cases by Using Importation Predictions, Volume 26, Number 7—July 2020 Research Pablo Martinez De Salazar1Comments to Author , René Niehus, Aimee Taylor1, Caroline O’Flaherty Buckee, and Marc LipsitchComments to Author Author affiliations: Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA Suggested citation for this article Abstract Cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 infection exported from mainland China could lead to self-sustained outbreaks in other countries. By February 2020, several countries were reporting imported SARS-CoV-2 cases. To contain the virus, early detection of imported SARS-CoV-2 cases is critical. We used air travel volume estimates from Wuhan, China, to international destinations and a generalized linear regression model to identify locations that could have undetected imported cases. Our model can be adjusted to account for exportation of cases from other locations as the virus spreads and more information on importations and transmission becomes available.",
"We used air travel volume estimates from Wuhan, China, to international destinations and a generalized linear regression model to identify locations that could have undetected imported cases. Our model can be adjusted to account for exportation of cases from other locations as the virus spreads and more information on importations and transmission becomes available. Early detection and appropriate control measures can reduce the risk for transmission in all locations. A novel coronavirus, later named severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 , was identified in December 2019 in the city of Wuhan, capital of Hubei Province, China, where cases were first confirmed .. During December 2019–February 2020, the number of confirmed cases increased drastically. Model estimates suggested that >75,000 persons were infected by January 25, 2020, and the epidemic had a doubling time of ≈6 days .. By the end of January 2020, travel restrictions were implemented for Wuhan and neighboring cities. Nonetheless, the virus spread from Wuhan to other cities in China and outside the country.",
"Model estimates suggested that >75,000 persons were infected by January 25, 2020, and the epidemic had a doubling time of ≈6 days .. By the end of January 2020, travel restrictions were implemented for Wuhan and neighboring cities. Nonetheless, the virus spread from Wuhan to other cities in China and outside the country. By February 4, 2020, a total of 23 locations outside mainland China reported cases, 22 of which reported imported cases; Spain reported a case caused by secondary transmission .. Most cases imported to other locations have been linked to recent travel history from China ., suggesting that air travel plays a major role in exportation of cases to locations outside of China. To prevent other cities and countries from becoming epicenters of the SARS-CoV-2 epidemic, substantial targeted public health interventions are required to detect cases and control local spread of the virus. We collected estimates of air travel volume from Wuhan to 194 international destinations.",
"To prevent other cities and countries from becoming epicenters of the SARS-CoV-2 epidemic, substantial targeted public health interventions are required to detect cases and control local spread of the virus. We collected estimates of air travel volume from Wuhan to 194 international destinations. We then identified 49 countries that had a score of >49.2/100 on category 2, Early Detection and Reporting of Epidemics of Potential International Concern, of the Global Health Security GHS Index .. We assumed these locations would be proficient at detecting SARS-CoV-2 and reporting confirmed imported cases, which we refer to as imported-and-reported cases. We ran a generalized linear regression model on this subset; based on the results, we generated predictions for the remainder of the sample. Using these predictions, we identified locations that might not be detecting imported cases. Methods To identify locations reporting fewer than predicted imported SARS-CoV-2 infected cases, we fit a model to data from 49 locations outside mainland China with high surveillance capacity according to the GHS Index ..",
"Using these predictions, we identified locations that might not be detecting imported cases. Methods To identify locations reporting fewer than predicted imported SARS-CoV-2 infected cases, we fit a model to data from 49 locations outside mainland China with high surveillance capacity according to the GHS Index .. Among these, 17 had high travel connectivity to Wuhan and 32 have low connectivity to Wuhan. We considered locations to be countries without any position on territorial claims. We performed a Poisson regression by using the cumulative number of imported-and-reported SARS-CoV-2 cases in these 49 countries and the estimated number of daily airline passengers from the Wuhan airport. We then compared predictions from this model with imported-and-reported cases across 194 locations from the GHS Index, excluding China as the epicenter of the outbreak.",
"We performed a Poisson regression by using the cumulative number of imported-and-reported SARS-CoV-2 cases in these 49 countries and the estimated number of daily airline passengers from the Wuhan airport. We then compared predictions from this model with imported-and-reported cases across 194 locations from the GHS Index, excluding China as the epicenter of the outbreak. The model requires data on imported-and-reported cases of SARS-CoV-2 infection, daily air travel volume, and surveillance capacity. We obtained data on imported-and-reported cases aggregated by destination from the World Health Organization technical report issued February 4, 2020 .. We assumed a case count of 0 for locations not listed. We used February 4 as the cutoff for cumulative imported-and-reported case counts because exported cases from Hubei Province dropped rapidly after this date ., likely because of travel restrictions for the province implement on January 23. We defined imported-and-reported cases as those with known travel history from China; of those, 83% had a travel history from Hubei Province and 17% traveled from unknown locations in China .. We excluded reported cases likely caused by transmission outside of China or cases in which the transmission source was still under investigation ..",
"We used February 4 as the cutoff for cumulative imported-and-reported case counts because exported cases from Hubei Province dropped rapidly after this date ., likely because of travel restrictions for the province implement on January 23. We defined imported-and-reported cases as those with known travel history from China; of those, 83% had a travel history from Hubei Province and 17% traveled from unknown locations in China .. We excluded reported cases likely caused by transmission outside of China or cases in which the transmission source was still under investigation .. In addition, we excluded Hong Kong, Macau, and Taiwan from our model because locally transmitted and imported cases were not disaggregated in these locations. We obtained data on daily air travel from a network-based modeling study S. Lai et al., unpub. data, Link that reported monthly air travel volume estimates for the 27 locations outside mainland China that are most connected to Wuhan. These estimates were calculated from International Air Travel Association data from February 2018, which includes direct and indirect flight itineraries from Wuhan.",
"data, Link that reported monthly air travel volume estimates for the 27 locations outside mainland China that are most connected to Wuhan. These estimates were calculated from International Air Travel Association data from February 2018, which includes direct and indirect flight itineraries from Wuhan. For these 27 locations, estimated air travel volumes are >6 passengers/day. We assumed that travel volumes for locations not among the most connected are censored by a detection limit. We used a common method of dealing with censored data from environmental sampling ., or metabolomics ., to set the daily air travel volume to half the minimum previously reported. Therefore, we used 3 passengers/day for estimated travel volumes for the 167 locations from the GHS Index not listed by Lai et al.",
"We used a common method of dealing with censored data from environmental sampling ., or metabolomics ., to set the daily air travel volume to half the minimum previously reported. Therefore, we used 3 passengers/day for estimated travel volumes for the 167 locations from the GHS Index not listed by Lai et al. We tested the robustness of our results by using a set of alternative values of 0.1, 1, and 6 passengers/day for the censored data. We defined high surveillance locations as those with a GHS Index for category 2 above the 75th quantile. We assessed the number of high surveillance locations, those with 0 imported-and-reported cases, and low surveillance locations, those with case counts >1 Table . For our model, we assumed that the cumulative imported-and-reported case counts across 49 high surveillance locations follow a Poisson distribution from the beginning of the epidemic until February 4, 2020.",
"We assessed the number of high surveillance locations, those with 0 imported-and-reported cases, and low surveillance locations, those with case counts >1 Table . For our model, we assumed that the cumulative imported-and-reported case counts across 49 high surveillance locations follow a Poisson distribution from the beginning of the epidemic until February 4, 2020. Then the expected case count is linearly proportional to the daily air travel volume in the following formula:where i denotes location, Ci denotes the imported-and-reported case count in a location, λi denotes the expected case count in a location, β denotes the regression coefficient, and xi denotes the daily air travel volume of a location. The Poisson model assumes cases are independent and that the variance is equal to the expected case count. Imported-and-reported cases likely meet the independence assumption because the value excludes cases with local transmission. We also checked the robustness of our results by using an over dispersed model with a negative binomial likelihood.",
"Imported-and-reported cases likely meet the independence assumption because the value excludes cases with local transmission. We also checked the robustness of our results by using an over dispersed model with a negative binomial likelihood. We computed the p value of the overdispersion parameter as shown in Gelman and Hill .. Thumbnail of Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in number of persons/day. No. cases refers to possible undetected imported SARS-CoV-2 cases. Solid line indicates the expected imported-and-reported case counts for locations. Dashed lines represent 95% prediction interval bounds smoothed for all locations.",
"Solid line indicates the expected imported-and-reported case counts for locations. Dashed lines represent 95% prediction interval bounds smoothed for all locations. Purple dots indicate location Figure 1. Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in number of... We used R version 3.6.1 Link to compute , the maximum likelihood estimate of β, and the expected imported-and-reported case count given high surveillance Figure 1 . We also computed the 95% prediction interval PI bounds under this model of high surveillance for all 194 values of daily air travel volume Figure 1 .",
"Air travel volume measured in number of... We used R version 3.6.1 Link to compute , the maximum likelihood estimate of β, and the expected imported-and-reported case count given high surveillance Figure 1 . We also computed the 95% prediction interval PI bounds under this model of high surveillance for all 194 values of daily air travel volume Figure 1 . First, we generated a bootstrapped dataset by sampling n locations with replacement among high surveillance locations. Then, we reestimated β by using the bootstrapped dataset. Finally, we simulated imported-and-reported case counts for all 194 locations under our model by using the estimate of β from the bootstrapped dataset. We repeated the 3 steps 50,000 times to generate 50,000 simulated imported-and-reported case counts for each of the locations computed to the lower and upper PI bounds PI 2.5%–97.5% .",
"Finally, we simulated imported-and-reported case counts for all 194 locations under our model by using the estimate of β from the bootstrapped dataset. We repeated the 3 steps 50,000 times to generate 50,000 simulated imported-and-reported case counts for each of the locations computed to the lower and upper PI bounds PI 2.5%–97.5% . We smoothed the 95% PI bounds by using ggplot2 in R .. We fit the imported-and-reported case counts of the 49 high surveillance locations to the model and plotted these alongside 145 locations with low surveillance capacity Figure 1 . We noted some overlap between high and low surveillance locations Figure 1 . Thumbnail of Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in number of persons/day.",
"Thumbnail of Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in number of persons/day. No. cases refers to possible undetected imported SARS-CoV-2 cases. Solid line shows the expected imported-and-reported case counts based on our model fitted to high surveillance locations, indicated by purple dots. Dashed lines indicate the 95% pr Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 .",
"Dashed lines indicate the 95% pr Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in... To assess the robustness of our results we ran 8 additional regression analyses by implementing a series of changes to the analysis. The changes included the following: set the daily air travel volume to 0.1, 1, or 6 passengers/day for locations not listed by Lai et al. unpub. data, Link Figure 2, panels A–C ; removed all locations not listed by Lai et al.",
"unpub. data, Link Figure 2, panels A–C ; removed all locations not listed by Lai et al. before fitting Figure 2, panel D ; defined high surveillance locations by using a more lenient GHS Index criterion, 50th quantile Figure 2, panel E , and a more stringent criterion, 95th quantile Figure 2, panel F ; excluded Thailand from the model because it is a high-leverage point Figure 2, panel G ; or used an overdispersed Poisson likelihood with a negative-binomial likelihood Figure 2, panel H . We provide code for these analyses on GitHub Link . Top Results We found that daily air travel volume positively correlates with imported-and-reported case counts of SARS-CoV-2 infection among high surveillance locations Figure 1 . We noted that increasing flight volume by 31 passengers/day is associated with 1 additional expected imported-and-reported case.",
"Top Results We found that daily air travel volume positively correlates with imported-and-reported case counts of SARS-CoV-2 infection among high surveillance locations Figure 1 . We noted that increasing flight volume by 31 passengers/day is associated with 1 additional expected imported-and-reported case. In addition, Singapore and India lie above the 95% PI in our model; Singapore had 12 more imported-and-reported cases 95% PI 6–17 cases than expected and India had 3 95% PI 1–3 cases more than expected. Thailand has a relatively high air travel volume compared with other locations, but it lies below the 95% PI, reporting 16 95% PI 1–40 cases fewer imported-and-reported cases than expected under the model. Indonesia lies below the PI and has no imported-and-reported cases, but the expected case count is 5 95% PI 1–10 cases in our model. Across all 8 robustness regression analyses, we consistently observed that Singapore lies above the 95% PI and Thailand and Indonesia lie below Figure 2 .",
"Indonesia lies below the PI and has no imported-and-reported cases, but the expected case count is 5 95% PI 1–10 cases in our model. Across all 8 robustness regression analyses, we consistently observed that Singapore lies above the 95% PI and Thailand and Indonesia lie below Figure 2 . India remains above the 95% PI in all robustness analyses except when we used the more stringent GHS Index, 95th quantile, for fitting; then India lies on the upper bound of the 95% PI Figure 2, panel F . Top Discussion We aimed to identify locations with likely undetected or underdetected imported cases of SARS-CoV-2 by fitting a model to the case counts in locations with high surveillance capacity and Wuhan-to-location air travel volumes. Our model can be adjusted to account for exportation of cases from locations other than Wuhan as the outbreak develops and more information on importations and self-sustained transmission becomes available. One key advantage of this model is that it does not rely on estimates of incidence or prevalence in the epicenter of the outbreak.",
"Our model can be adjusted to account for exportation of cases from locations other than Wuhan as the outbreak develops and more information on importations and self-sustained transmission becomes available. One key advantage of this model is that it does not rely on estimates of incidence or prevalence in the epicenter of the outbreak. Also, we intentionally used a simple generalized linear model. The linearity of the expected case count means that we have only 1 regression coefficient in the model and no extra parameters. The Poisson likelihood then captures the many 0-counts observed for less highly connected locations but also describes the slope between case-count and flight data among more connected locations. We believe this model provides the most parsimonious phenomenologic description of the data.",
"The Poisson likelihood then captures the many 0-counts observed for less highly connected locations but also describes the slope between case-count and flight data among more connected locations. We believe this model provides the most parsimonious phenomenologic description of the data. According to our model, locations above the 95% PI of imported-and-reported cases could have higher case-detection capacity. Locations below the 95% PI might have undetected cases because of expected imported-and-reported case counts under high surveillance. Underdetection of cases could increase the international spread of the outbreak because the transmission chain could be lost, reducing opportunities to deploy case-based control strategies. We recommend rapid strengthening of outbreak surveillance and control efforts in locations below the 95% PI lower bound, particularly Indonesia, to curb potential local transmission.",
"Underdetection of cases could increase the international spread of the outbreak because the transmission chain could be lost, reducing opportunities to deploy case-based control strategies. We recommend rapid strengthening of outbreak surveillance and control efforts in locations below the 95% PI lower bound, particularly Indonesia, to curb potential local transmission. Early detection of cases and implantation of appropriate control measures can reduce the risk for self-sustained transmission in all locations. Top Dr. De Salazar is a research fellow at Harvard T.H. Chan School of Public Health, working on multiscale statistical models of infectious diseases within host, population, and metapopulation models. His research interests include diagnostic laboratory methods and public health response.",
"Chan School of Public Health, working on multiscale statistical models of infectious diseases within host, population, and metapopulation models. His research interests include diagnostic laboratory methods and public health response. Top Acknowledgments We thank Pamela Martinez, Nicholas Jewel, and Stephen Kissler for valuable feedback. This work was supported by US National Institute of General Medical Sciences award no. U54GM088558 . P.M.D was supported by the Fellowship Foundation Ramon Areces. A.R.T. and C.O.B. were supported by a Maximizing Investigator’s Research Award no. R35GM124715-02 from the US National Institute of General Medical Sciences.",
"A.R.T. and C.O.B. were supported by a Maximizing Investigator’s Research Award no. R35GM124715-02 from the US National Institute of General Medical Sciences. The authors are solely responsible for this content and it does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. Declaration of interests: Marc Lipsitch has received consulting fees from Merck. All other authors declare no competing interests. Top References Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–3.",
"A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–3. Wu JT, Leung K, Leung GM. Nowcasting and forecasting the potential domestic and international spread of the 2019-nCoV outbreak originating in Wuhan, China: a modelling study. Lancet. 2020;395:689–97. DOIExternal LinkPubMedExternal Link World Health Organization. Coronavirus disease 2019 COVID-19 situation report—15, 4 Feb 2020 cited 2020 Feb 14 . Link Nuclear Threat Initiative and Johns Hopkins Center for Health Security. Global health security index cited 2020 Feb 14 . Link US Environmental Protection Agency. Data quality assessment: statistical methods for practitioners EPA QA/G9-S cited 2020 Feb 14 .",
"Global health security index cited 2020 Feb 14 . Link US Environmental Protection Agency. Data quality assessment: statistical methods for practitioners EPA QA/G9-S cited 2020 Feb 14 . Washington: The Agency; 2006. Link Lamichhane S, Sen P, Dickens AM, Hyötyläinen T, Orešič M. An overview of metabolomics data analysis: current tools and future perspectives. In: Jaumot J, Bedia C, Tauler R, editors. Comprehensive analytical chemistry. Vol. 82. Amsterdam: Elsevier; 2018. p. 387–413. Gelman A, Hill J. Analytical methods for social research. In: Data analysis using regression and multilevel/hierarchical models. Cambridge: Cambridge University Press; 2006. p. 235–236.",
"Gelman A, Hill J. Analytical methods for social research. In: Data analysis using regression and multilevel/hierarchical models. Cambridge: Cambridge University Press; 2006. p. 235–236. Wickham H. ggplot2: elegant graphics for data analysis. New York: Springer; 2016. Top Figures Figure 1. Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in... Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume... Table Table.",
"Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume... Table Table. Surveillance capacity of locations with and without imported-and-reported cases of severe acute respiratory syndrome coronavirus 2, 2020 Top Suggested citation for this article: De Salazar PM, Niehus R, Taylor A, O’Flaherty Buckee C, Lipsitch M. Identifying locations with possible undetected imported severe acute respiratory syndrome coronavirus 2 cases by using importation predictions. Emerg Infect Dis. 2020 Jul date cited . DOI: 10.3201/eid2607.200250 Original Publication Date: 3/24/2020 1These authors contributed equally to this article.",
"Emerg Infect Dis. 2020 Jul date cited . DOI: 10.3201/eid2607.200250 Original Publication Date: 3/24/2020 1These authors contributed equally to this article. Table of Contents – Volume 26, Number 7—July 2020"
] | 186 | 247 |
What is the doubling time of the COVID-19 pandemic? | ≈6 days | [
"Identifying Locations with Possible Undetected Imported Severe Acute Respiratory Syndrome Coronavirus 2 Cases by Using Importation Predictions, Volume 26, Number 7—July 2020 Research Pablo Martinez De Salazar1Comments to Author , René Niehus, Aimee Taylor1, Caroline O’Flaherty Buckee, and Marc LipsitchComments to Author Author affiliations: Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA Suggested citation for this article Abstract Cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 infection exported from mainland China could lead to self-sustained outbreaks in other countries. By February 2020, several countries were reporting imported SARS-CoV-2 cases. To contain the virus, early detection of imported SARS-CoV-2 cases is critical. We used air travel volume estimates from Wuhan, China, to international destinations and a generalized linear regression model to identify locations that could have undetected imported cases. Our model can be adjusted to account for exportation of cases from other locations as the virus spreads and more information on importations and transmission becomes available.",
"We used air travel volume estimates from Wuhan, China, to international destinations and a generalized linear regression model to identify locations that could have undetected imported cases. Our model can be adjusted to account for exportation of cases from other locations as the virus spreads and more information on importations and transmission becomes available. Early detection and appropriate control measures can reduce the risk for transmission in all locations. A novel coronavirus, later named severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 , was identified in December 2019 in the city of Wuhan, capital of Hubei Province, China, where cases were first confirmed .. During December 2019–February 2020, the number of confirmed cases increased drastically. Model estimates suggested that >75,000 persons were infected by January 25, 2020, and the epidemic had a doubling time of ≈6 days .. By the end of January 2020, travel restrictions were implemented for Wuhan and neighboring cities. Nonetheless, the virus spread from Wuhan to other cities in China and outside the country.",
"Model estimates suggested that >75,000 persons were infected by January 25, 2020, and the epidemic had a doubling time of ≈6 days .. By the end of January 2020, travel restrictions were implemented for Wuhan and neighboring cities. Nonetheless, the virus spread from Wuhan to other cities in China and outside the country. By February 4, 2020, a total of 23 locations outside mainland China reported cases, 22 of which reported imported cases; Spain reported a case caused by secondary transmission .. Most cases imported to other locations have been linked to recent travel history from China ., suggesting that air travel plays a major role in exportation of cases to locations outside of China. To prevent other cities and countries from becoming epicenters of the SARS-CoV-2 epidemic, substantial targeted public health interventions are required to detect cases and control local spread of the virus. We collected estimates of air travel volume from Wuhan to 194 international destinations.",
"To prevent other cities and countries from becoming epicenters of the SARS-CoV-2 epidemic, substantial targeted public health interventions are required to detect cases and control local spread of the virus. We collected estimates of air travel volume from Wuhan to 194 international destinations. We then identified 49 countries that had a score of >49.2/100 on category 2, Early Detection and Reporting of Epidemics of Potential International Concern, of the Global Health Security GHS Index .. We assumed these locations would be proficient at detecting SARS-CoV-2 and reporting confirmed imported cases, which we refer to as imported-and-reported cases. We ran a generalized linear regression model on this subset; based on the results, we generated predictions for the remainder of the sample. Using these predictions, we identified locations that might not be detecting imported cases. Methods To identify locations reporting fewer than predicted imported SARS-CoV-2 infected cases, we fit a model to data from 49 locations outside mainland China with high surveillance capacity according to the GHS Index ..",
"Using these predictions, we identified locations that might not be detecting imported cases. Methods To identify locations reporting fewer than predicted imported SARS-CoV-2 infected cases, we fit a model to data from 49 locations outside mainland China with high surveillance capacity according to the GHS Index .. Among these, 17 had high travel connectivity to Wuhan and 32 have low connectivity to Wuhan. We considered locations to be countries without any position on territorial claims. We performed a Poisson regression by using the cumulative number of imported-and-reported SARS-CoV-2 cases in these 49 countries and the estimated number of daily airline passengers from the Wuhan airport. We then compared predictions from this model with imported-and-reported cases across 194 locations from the GHS Index, excluding China as the epicenter of the outbreak.",
"We performed a Poisson regression by using the cumulative number of imported-and-reported SARS-CoV-2 cases in these 49 countries and the estimated number of daily airline passengers from the Wuhan airport. We then compared predictions from this model with imported-and-reported cases across 194 locations from the GHS Index, excluding China as the epicenter of the outbreak. The model requires data on imported-and-reported cases of SARS-CoV-2 infection, daily air travel volume, and surveillance capacity. We obtained data on imported-and-reported cases aggregated by destination from the World Health Organization technical report issued February 4, 2020 .. We assumed a case count of 0 for locations not listed. We used February 4 as the cutoff for cumulative imported-and-reported case counts because exported cases from Hubei Province dropped rapidly after this date ., likely because of travel restrictions for the province implement on January 23. We defined imported-and-reported cases as those with known travel history from China; of those, 83% had a travel history from Hubei Province and 17% traveled from unknown locations in China .. We excluded reported cases likely caused by transmission outside of China or cases in which the transmission source was still under investigation ..",
"We used February 4 as the cutoff for cumulative imported-and-reported case counts because exported cases from Hubei Province dropped rapidly after this date ., likely because of travel restrictions for the province implement on January 23. We defined imported-and-reported cases as those with known travel history from China; of those, 83% had a travel history from Hubei Province and 17% traveled from unknown locations in China .. We excluded reported cases likely caused by transmission outside of China or cases in which the transmission source was still under investigation .. In addition, we excluded Hong Kong, Macau, and Taiwan from our model because locally transmitted and imported cases were not disaggregated in these locations. We obtained data on daily air travel from a network-based modeling study S. Lai et al., unpub. data, Link that reported monthly air travel volume estimates for the 27 locations outside mainland China that are most connected to Wuhan. These estimates were calculated from International Air Travel Association data from February 2018, which includes direct and indirect flight itineraries from Wuhan.",
"data, Link that reported monthly air travel volume estimates for the 27 locations outside mainland China that are most connected to Wuhan. These estimates were calculated from International Air Travel Association data from February 2018, which includes direct and indirect flight itineraries from Wuhan. For these 27 locations, estimated air travel volumes are >6 passengers/day. We assumed that travel volumes for locations not among the most connected are censored by a detection limit. We used a common method of dealing with censored data from environmental sampling ., or metabolomics ., to set the daily air travel volume to half the minimum previously reported. Therefore, we used 3 passengers/day for estimated travel volumes for the 167 locations from the GHS Index not listed by Lai et al.",
"We used a common method of dealing with censored data from environmental sampling ., or metabolomics ., to set the daily air travel volume to half the minimum previously reported. Therefore, we used 3 passengers/day for estimated travel volumes for the 167 locations from the GHS Index not listed by Lai et al. We tested the robustness of our results by using a set of alternative values of 0.1, 1, and 6 passengers/day for the censored data. We defined high surveillance locations as those with a GHS Index for category 2 above the 75th quantile. We assessed the number of high surveillance locations, those with 0 imported-and-reported cases, and low surveillance locations, those with case counts >1 Table . For our model, we assumed that the cumulative imported-and-reported case counts across 49 high surveillance locations follow a Poisson distribution from the beginning of the epidemic until February 4, 2020.",
"We assessed the number of high surveillance locations, those with 0 imported-and-reported cases, and low surveillance locations, those with case counts >1 Table . For our model, we assumed that the cumulative imported-and-reported case counts across 49 high surveillance locations follow a Poisson distribution from the beginning of the epidemic until February 4, 2020. Then the expected case count is linearly proportional to the daily air travel volume in the following formula:where i denotes location, Ci denotes the imported-and-reported case count in a location, λi denotes the expected case count in a location, β denotes the regression coefficient, and xi denotes the daily air travel volume of a location. The Poisson model assumes cases are independent and that the variance is equal to the expected case count. Imported-and-reported cases likely meet the independence assumption because the value excludes cases with local transmission. We also checked the robustness of our results by using an over dispersed model with a negative binomial likelihood.",
"Imported-and-reported cases likely meet the independence assumption because the value excludes cases with local transmission. We also checked the robustness of our results by using an over dispersed model with a negative binomial likelihood. We computed the p value of the overdispersion parameter as shown in Gelman and Hill .. Thumbnail of Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in number of persons/day. No. cases refers to possible undetected imported SARS-CoV-2 cases. Solid line indicates the expected imported-and-reported case counts for locations. Dashed lines represent 95% prediction interval bounds smoothed for all locations.",
"Solid line indicates the expected imported-and-reported case counts for locations. Dashed lines represent 95% prediction interval bounds smoothed for all locations. Purple dots indicate location Figure 1. Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in number of... We used R version 3.6.1 Link to compute , the maximum likelihood estimate of β, and the expected imported-and-reported case count given high surveillance Figure 1 . We also computed the 95% prediction interval PI bounds under this model of high surveillance for all 194 values of daily air travel volume Figure 1 .",
"Air travel volume measured in number of... We used R version 3.6.1 Link to compute , the maximum likelihood estimate of β, and the expected imported-and-reported case count given high surveillance Figure 1 . We also computed the 95% prediction interval PI bounds under this model of high surveillance for all 194 values of daily air travel volume Figure 1 . First, we generated a bootstrapped dataset by sampling n locations with replacement among high surveillance locations. Then, we reestimated β by using the bootstrapped dataset. Finally, we simulated imported-and-reported case counts for all 194 locations under our model by using the estimate of β from the bootstrapped dataset. We repeated the 3 steps 50,000 times to generate 50,000 simulated imported-and-reported case counts for each of the locations computed to the lower and upper PI bounds PI 2.5%–97.5% .",
"Finally, we simulated imported-and-reported case counts for all 194 locations under our model by using the estimate of β from the bootstrapped dataset. We repeated the 3 steps 50,000 times to generate 50,000 simulated imported-and-reported case counts for each of the locations computed to the lower and upper PI bounds PI 2.5%–97.5% . We smoothed the 95% PI bounds by using ggplot2 in R .. We fit the imported-and-reported case counts of the 49 high surveillance locations to the model and plotted these alongside 145 locations with low surveillance capacity Figure 1 . We noted some overlap between high and low surveillance locations Figure 1 . Thumbnail of Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in number of persons/day.",
"Thumbnail of Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in number of persons/day. No. cases refers to possible undetected imported SARS-CoV-2 cases. Solid line shows the expected imported-and-reported case counts based on our model fitted to high surveillance locations, indicated by purple dots. Dashed lines indicate the 95% pr Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 .",
"Dashed lines indicate the 95% pr Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume measured in... To assess the robustness of our results we ran 8 additional regression analyses by implementing a series of changes to the analysis. The changes included the following: set the daily air travel volume to 0.1, 1, or 6 passengers/day for locations not listed by Lai et al. unpub. data, Link Figure 2, panels A–C ; removed all locations not listed by Lai et al.",
"unpub. data, Link Figure 2, panels A–C ; removed all locations not listed by Lai et al. before fitting Figure 2, panel D ; defined high surveillance locations by using a more lenient GHS Index criterion, 50th quantile Figure 2, panel E , and a more stringent criterion, 95th quantile Figure 2, panel F ; excluded Thailand from the model because it is a high-leverage point Figure 2, panel G ; or used an overdispersed Poisson likelihood with a negative-binomial likelihood Figure 2, panel H . We provide code for these analyses on GitHub Link . Top Results We found that daily air travel volume positively correlates with imported-and-reported case counts of SARS-CoV-2 infection among high surveillance locations Figure 1 . We noted that increasing flight volume by 31 passengers/day is associated with 1 additional expected imported-and-reported case.",
"Top Results We found that daily air travel volume positively correlates with imported-and-reported case counts of SARS-CoV-2 infection among high surveillance locations Figure 1 . We noted that increasing flight volume by 31 passengers/day is associated with 1 additional expected imported-and-reported case. In addition, Singapore and India lie above the 95% PI in our model; Singapore had 12 more imported-and-reported cases 95% PI 6–17 cases than expected and India had 3 95% PI 1–3 cases more than expected. Thailand has a relatively high air travel volume compared with other locations, but it lies below the 95% PI, reporting 16 95% PI 1–40 cases fewer imported-and-reported cases than expected under the model. Indonesia lies below the PI and has no imported-and-reported cases, but the expected case count is 5 95% PI 1–10 cases in our model. Across all 8 robustness regression analyses, we consistently observed that Singapore lies above the 95% PI and Thailand and Indonesia lie below Figure 2 .",
"Indonesia lies below the PI and has no imported-and-reported cases, but the expected case count is 5 95% PI 1–10 cases in our model. Across all 8 robustness regression analyses, we consistently observed that Singapore lies above the 95% PI and Thailand and Indonesia lie below Figure 2 . India remains above the 95% PI in all robustness analyses except when we used the more stringent GHS Index, 95th quantile, for fitting; then India lies on the upper bound of the 95% PI Figure 2, panel F . Top Discussion We aimed to identify locations with likely undetected or underdetected imported cases of SARS-CoV-2 by fitting a model to the case counts in locations with high surveillance capacity and Wuhan-to-location air travel volumes. Our model can be adjusted to account for exportation of cases from locations other than Wuhan as the outbreak develops and more information on importations and self-sustained transmission becomes available. One key advantage of this model is that it does not rely on estimates of incidence or prevalence in the epicenter of the outbreak.",
"Our model can be adjusted to account for exportation of cases from locations other than Wuhan as the outbreak develops and more information on importations and self-sustained transmission becomes available. One key advantage of this model is that it does not rely on estimates of incidence or prevalence in the epicenter of the outbreak. Also, we intentionally used a simple generalized linear model. The linearity of the expected case count means that we have only 1 regression coefficient in the model and no extra parameters. The Poisson likelihood then captures the many 0-counts observed for less highly connected locations but also describes the slope between case-count and flight data among more connected locations. We believe this model provides the most parsimonious phenomenologic description of the data.",
"The Poisson likelihood then captures the many 0-counts observed for less highly connected locations but also describes the slope between case-count and flight data among more connected locations. We believe this model provides the most parsimonious phenomenologic description of the data. According to our model, locations above the 95% PI of imported-and-reported cases could have higher case-detection capacity. Locations below the 95% PI might have undetected cases because of expected imported-and-reported case counts under high surveillance. Underdetection of cases could increase the international spread of the outbreak because the transmission chain could be lost, reducing opportunities to deploy case-based control strategies. We recommend rapid strengthening of outbreak surveillance and control efforts in locations below the 95% PI lower bound, particularly Indonesia, to curb potential local transmission.",
"Underdetection of cases could increase the international spread of the outbreak because the transmission chain could be lost, reducing opportunities to deploy case-based control strategies. We recommend rapid strengthening of outbreak surveillance and control efforts in locations below the 95% PI lower bound, particularly Indonesia, to curb potential local transmission. Early detection of cases and implantation of appropriate control measures can reduce the risk for self-sustained transmission in all locations. Top Dr. De Salazar is a research fellow at Harvard T.H. Chan School of Public Health, working on multiscale statistical models of infectious diseases within host, population, and metapopulation models. His research interests include diagnostic laboratory methods and public health response.",
"Chan School of Public Health, working on multiscale statistical models of infectious diseases within host, population, and metapopulation models. His research interests include diagnostic laboratory methods and public health response. Top Acknowledgments We thank Pamela Martinez, Nicholas Jewel, and Stephen Kissler for valuable feedback. This work was supported by US National Institute of General Medical Sciences award no. U54GM088558 . P.M.D was supported by the Fellowship Foundation Ramon Areces. A.R.T. and C.O.B. were supported by a Maximizing Investigator’s Research Award no. R35GM124715-02 from the US National Institute of General Medical Sciences.",
"A.R.T. and C.O.B. were supported by a Maximizing Investigator’s Research Award no. R35GM124715-02 from the US National Institute of General Medical Sciences. The authors are solely responsible for this content and it does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. Declaration of interests: Marc Lipsitch has received consulting fees from Merck. All other authors declare no competing interests. Top References Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–3.",
"A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–3. Wu JT, Leung K, Leung GM. Nowcasting and forecasting the potential domestic and international spread of the 2019-nCoV outbreak originating in Wuhan, China: a modelling study. Lancet. 2020;395:689–97. DOIExternal LinkPubMedExternal Link World Health Organization. Coronavirus disease 2019 COVID-19 situation report—15, 4 Feb 2020 cited 2020 Feb 14 . Link Nuclear Threat Initiative and Johns Hopkins Center for Health Security. Global health security index cited 2020 Feb 14 . Link US Environmental Protection Agency. Data quality assessment: statistical methods for practitioners EPA QA/G9-S cited 2020 Feb 14 .",
"Global health security index cited 2020 Feb 14 . Link US Environmental Protection Agency. Data quality assessment: statistical methods for practitioners EPA QA/G9-S cited 2020 Feb 14 . Washington: The Agency; 2006. Link Lamichhane S, Sen P, Dickens AM, Hyötyläinen T, Orešič M. An overview of metabolomics data analysis: current tools and future perspectives. In: Jaumot J, Bedia C, Tauler R, editors. Comprehensive analytical chemistry. Vol. 82. Amsterdam: Elsevier; 2018. p. 387–413. Gelman A, Hill J. Analytical methods for social research. In: Data analysis using regression and multilevel/hierarchical models. Cambridge: Cambridge University Press; 2006. p. 235–236.",
"Gelman A, Hill J. Analytical methods for social research. In: Data analysis using regression and multilevel/hierarchical models. Cambridge: Cambridge University Press; 2006. p. 235–236. Wickham H. ggplot2: elegant graphics for data analysis. New York: Springer; 2016. Top Figures Figure 1. Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 by air travel volume from Wuhan, China. Air travel volume measured in... Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume... Table Table.",
"Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 SARS-CoV-2 . Air travel volume... Table Table. Surveillance capacity of locations with and without imported-and-reported cases of severe acute respiratory syndrome coronavirus 2, 2020 Top Suggested citation for this article: De Salazar PM, Niehus R, Taylor A, O’Flaherty Buckee C, Lipsitch M. Identifying locations with possible undetected imported severe acute respiratory syndrome coronavirus 2 cases by using importation predictions. Emerg Infect Dis. 2020 Jul date cited . DOI: 10.3201/eid2607.200250 Original Publication Date: 3/24/2020 1These authors contributed equally to this article.",
"Emerg Infect Dis. 2020 Jul date cited . DOI: 10.3201/eid2607.200250 Original Publication Date: 3/24/2020 1These authors contributed equally to this article. Table of Contents – Volume 26, Number 7—July 2020"
] | 186 | 272 |
When did the first known cases of Middle East respiratory syndrome (MERS) occur? | in 2012 | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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Where did the first known cases of Middle East respiratory syndrome (MERS) occur? | in Jordan | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,174 |
Where was the the case first to be publicly reported was from ? | Jeddah, in the Kingdom of Saudi Arabia (KSA) | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,175 |
In what animals MERS-CoV sequences have been found ? | in a bat and in many dromedary camels (DC). | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,176 |
Where is MERS-CoV is enzootic in DC? | across the Arabian Peninsula and in parts of Africa | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What does MERS-COV cause? | mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,178 |
Precisely how does the virus transmit to humans? | remains unknown | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What appears to be a requirement for transmission? | close and lengthy exposure | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is a focal point of MERS? | The KSA | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is MERS mostly known as? | lower respiratory tract (LRT) disease | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,182 |
What does the MERS LRT disease involve? | fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,183 |
Where else MERS-COV has also been detected? | in mild and influenza-like illnesses and in those with no signs or symptoms. | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,184 |
Who suffers severe diseases from MERS? | Older males | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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Compared to severe acute respiratory syndrome (SARS) and another sometimes- fatal zoonotic coronavirus disease, how does MERS affect the patients? | MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,186 |
To what have most human cases of MERS been linked? | to lapses in infection prevention and control (IPC) in healthcare settings | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,187 |
Among whom 20% of the virus detection are reported? | among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What have sero-surveys of MERS virus found? | widespread evidence of past infection in adult camels and limited past exposure among humans. | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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How was the first culture of the new Coronavirus announced? | An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia (KSA) | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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Where was the Email published? | on the website of the professional emerging diseases (ProMED) network | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,191 |
When was the Email published? | 20 th September 2012 | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,192 |
Whose was the first reported case? | a 60 year old man from Bisha in the KSA | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,193 |
How many viral RNA or virus-specific antibodies been detected? | 1,493 | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,194 |
What is the death rate from MERS-COV? | over a third of the positive people dying (at least 527, 35 %) | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What did the discovery process over two to three years reveal? | a virus that had infected over 90 % of adult dromedary camels (DC; Camelus dromedarius) in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What does subsequent transmission of MERS-CoV to other humans require? | relatively close and prolonged exposure | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What would restrict access to both the virus and to viral diagnostics ? | The first viral isolate was patented | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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How was the virus then made freely available? | sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics were quickly described | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What has epidemiology and research identified the MERS-CoV's cell receptor is? | as exopeptidase dipeptidyl peptidase 4 (DPP4; also called CD26) | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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How does MERS-CoV compare with SARS-CoV? | eliciting a more proinflammatory response than SARS-CoV | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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How does MERS-CoV compare with SARS-CoV? | MERS kills its human host more often than SARS did (20-40 % versus 9 % for SARS | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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How does MERS-CoV spread among people? | From intermittent animal-to-human spill-over events | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,205 |
Who gets more severe disease from MERS? | among older adults, especially males, with pre-existing diseases. | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is the spread of MERS-CoV among humans, associated with? | with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (HCWs). | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What do the DCs suffer with MERS-CoV infection? | the equivalent of a 'common cold' from MERS-CoV infection, | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What happens to humans infected by MERS-CoV virus? | the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,209 |
What is the incubation period of MERS? | five to six days, ranging from two to 16 days | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,210 |
What is the duration between when illness begins in one person and subsequently spreads to another? | 13 to 14 days | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,211 |
What is the median time to death in case of progressive illness? | 11 to 13 days | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is the progression of symptoms to disease? | Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,214 |
What did the later WHO definition of MERS clearly state? | a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,216 |
What percentage of cases KSA has been a source of? | 79 % | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,217 |
What is severe MARS noted for? | its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What symptoms appear among the confirmed cases of MERS? | fever, cough and upper respiratory tract (URT) signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What do patients often present to a hospital with, in cases of MERS? | pneumonia | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What can MERS disease progress to? | acute respiratory distress syndrome and multiorgan system failure | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,224 |
What percentage of all reported cases has MERS reportedly killed ? | 35 % | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,225 |
What percentage of of all reported cases has MERS reportedly killed in KSA? | 42 % | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What was mortality in South Korea from MERS disease? | ranged from 7 % among younger age groups to 40 % among those aged 60 years and above | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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Which are the preferred method for MERS-CoV detection? | PCR-based techniques | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What have become a key diagnostic and taxonomic target for CoV species identification? | The first open reading frames | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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Why it can be concluded that MERS-COV is a novel and distinct virus? | less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5 | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What indicates the likely presence of infectious virus? | Detection of viral proteins rather than viral RNA | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is the sensitivity with which immunochromatographic tool could detect recombinant MERS-CoV nucleocapsid protein? | 94 % | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is the specificity with which immunochromatographic tool could detect recombinant MERS-CoV nucleocapsid protein? | 100 % | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is a different approach for the detection? | monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is usual in serology testing? | to detect a viral footprint, in the form of antibodies | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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How have some sero-assays bypassed the risks of working with infectious virus? | by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins [ | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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How have some sero-assays bypassed the risks of working with infectious virus? | using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What is the detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by? | by confirmatory IFA and/ or a plaque-reduction neutralization (PRNT) or MNT test. | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,240 |
What does the confirmatory process aim to ensure? | the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs (bovine CoV, BCoV) or humans (HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV). | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,241 |
When does generally MERS infection does not trigger a detectable immune response? | if only mild disease or asymptomatic infection results | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,242 |
When does the WHO recommend samlinf from the LRT? | when sample collection is delayed by a week or more after onset of symptoms | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What do the recommended samples include? | are recommended when URT sampling is to be conducted | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What are recommended when URT sampling is to be conducted? | an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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When is a single sample suggested to be sufficient? | Human urine and stool have been found to contain MERS-CoV RNA | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,247 |
How long human urine and stool have been found to contain MERS-CoV RNA? | 12 to 26 days after symptom onset | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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What do individual studies report on viral shedding? | long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,249 |
What fraction of MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum) ? | Over three quarters of | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,262 |
How long MERS cases shed viral RNA in their LRT specimens (tracheal aspirates and sputum)? | at least 30 days | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,263 |
What percentage of contacts were still shedding RNA in their URT specimens? | 30 % | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
] | 1,741 | 4,264 |
What samples returned the highest MERS viral load values? | the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or | [
"The first known cases of Middle East respiratory syndrome MERS , associated with infection by a novel coronavirus CoV , occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia KSA . Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels DC . MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases.",
"Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract LRT disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal.",
"Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome SARS , another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury it also has an affinity for growth in kidney cells under laboratory conditions , is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control IPC in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers HCWs and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.",
"Sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article .1186/s12985-015-0439-5 contains supplementary material, which is available to authorized users. Text: An email from Dr Ali Mohamed Zaki, an Egyptian virologist working at the Dr Soliman Fakeeh Hospital in Jeddah in the Kingdom of Saudi Arabia KSA announced the first culture of a new coronavirus to the world. The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA.",
"The email was published on the website of the professional emerging diseases ProMED network on 20 th September 2012 Fig. 1 and described the first reported case, a 60 year old man from Bisha in the KSA. This information led to the rapid discovery of a second case of the virus, this time in an ill patient in the United Kingdom, who had been transferred from Qatar for care . The new virus was initially called novel coronavirus nCoV and subsequentlty entitled the Middle East respiratoy syndrome coronavirus MERS-CoV . As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA .",
"As of 2 nd of September 2015, there have been 1,493 detections of viral RNA or virus-specific antibodies across 26 countries Additional file 1: Figure S1 confirmed by the World Health Organization WHO , with over a third of the positive people dying at least 527, 35 % . Since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels DC; Camelus dromedarius in the KSA , also DCs across the Arabian Peninsula and parts of Africa that are a source of DC imports for the KSA . To date, MERS-CoV has not been detected in DCs tested in zoos or herds from other parts of the world . Occasionally, virus is transmitted from infected DCs to exposed humans. Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics .",
"Subsequent transmission to other humans requires relatively close and prolonged exposure . The first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics . However, sensitive, validated reverse transcriptase real-time polymerase chain reaction RT-rtPCR -based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations . Subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 DPP4; also called CD26 ; that MERS-CoV has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than SARS-CoV; is widespread in DCs; has the potential to infect other animals and that MERS kills its human host more often than SARS did 20-40 % versus 9 % for SARS . In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases.",
"In humans, overt disease was given the name Middle East respiratory syndrome, with the acronym MERS. From intermittent animal-to-human spill-over events, the MERS-CoV spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. The spread of MERS-CoV among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers HCWs . Although DCs appear to suffer the equivalent of a 'common cold' from MERS-CoV infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another .",
"It has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans . Studies have established that the mean incubation period for MERS is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another . Among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days . Fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome . The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula.",
"The first WHO case definition defined probable cases of MERS based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract LRT involvement. It also included roles for contact with a probable or confirmed case or for travel or residence within the Arabian Peninsula. If strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on . From July 2013, the revised WHO case definition included the importance of seeking out and understanding the role of asymptomatic cases and from June 2014, the WHO definition more clearly stated that a confirmed case included any person whose sample was RT-PCR positive for MERS-CoV, or who produced a seroconversion, irrespective of clinical signs and symptoms. Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation .",
"Apart from the WHO and the KSA Ministry of Health reports, asymptomatic or subclinical cases of MERS-CoV infection were documented in the scientific literature although not always as often as occurred early on . The KSA definition of a case became more strict on 13 th May 2014, relying on the presence of both clinical features and laboratory confirmation . Testing of asymptomatic people was recommended against from December 2014 , reinforced by a case definition released by the KSA Ministry of Health in June 2015 . The KSA has been the source of 79 % of human cases. Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution .",
"Severe MERS is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions . Interestingly in June 2015, an outbreak in South Korea followed a similar distribution . Among laboratory confirmed cases, fever, cough and upper respiratory tract URT signs and symptoms usually occur first, followed within a week by progressive LRT distress and lymphopaenia . Patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported . Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported .",
"Disease can progress to acute respiratory distress syndrome and multiorgan system failure . MERS has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the KSA, yet only 19 % of cases in South Korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported . General supportive care is key to managing severe cases . Children under the age of 14 years are rarely reported to be positive for MERS-CoV, comprising only 1.1 % n = 16 of total reported cases. Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa .",
"Between 1 st September 2012 and 2 nd December 2013, a study described the then tally of paediatric cases in the KSA, which stood at 11 two to 16 years of age; median 13 years ; nine were asymptomatic 72 % and one infant died . In Amman, Jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for MERS-CoV RNA, despite being collected at a similar time to the first known outbreak of MERS-CoV in the neighbouring town of Al-Zarqa . A second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not RT-rtPCR positive, the mother did subsequently develop antibodies to MERS-CoV, suggestive of recent infection . Her exposure history to a MERS-CoV RT-rtPCR positive relative and an antibody-reactive husband, her incubation period and her symptom history met the WHO criteria for being a probable MERS-CoV case . Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells .",
"Diagnostic methods were published within days of the ProMED email announcing the first MERS case , including several now gold standard in-house RT-rtPCR assays Fig. 2 as well as virus culture in Vero and LLC-MK2 cells . A colorectal adenocarcinoma Caco-2 epithelial cell line has since been recommended for isolation of infections MERS-CoV . We previously . . Open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions UTR; grey rectangles . FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator.",
"FS-frame-shift. Predicted regions encompassing recombination break-points are indicated by orange pills. Created using Geneious v8.1 and annotated using Adobe Illustrator. Beneath this is a schematic depicting the location of RT-PCR primers blue arrows indicate direction and oligoprobes green rectangles used in the earliest RT-rtPCR screening assays and conventional, semi-nested three primers RT-PCR confirmatory sequencing assays . Publication order is noted by first 27 th September 2012; red and second 6 th December 2012; orange coloured rectangles; both from Corman et al. Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants.",
"Those assays recommended by the WHO are highlighted underneath by yellow dots . The NSeq reverse primer has consistently contained one sequence mismatch with some MERS-CoV variants. An altered version of that from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier reviewed the broad tropism of MERS-CoV . However, as is well described, cell culture is a slow, specialised and insensitive method while PCR-based techniques are the preferred method for MERS-CoV detection. The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification.",
"The first open reading frames ORF 1a and 1b; Fig. 2 have become a key diagnostic and taxonomic target for CoV species identification. With less than 80 % identity between the amino acid sequence of MERS ORF 1ab and betacoronavirus relatives, Tylonycteris bat HKU4 and Pipistrellus bat HKU5, it can be concluded that it is a novel and distinct virus. MERS-CoV is predicted to encode ten open reading frames with 5' and 3' untranslated regions . The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins.",
"The structural proteins include the spike S , envelope E , membrane M and nucleocapsid N . The products of ORF1a and ORF1b are predicted to encode nonstructural proteins. The majority of specimen testing to date has employed validated RT-rtPCR assays shown to be sensitive and specific . The RealStar® kit uses these WHOrecommended assays . The target sequences of these screening assays have not changed among genomes examined until at least mid-2015 IMM observation . Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment.",
"Other RT-rtPCR assays have been developed and validated for use as laboratory-based diagnostic tools . Additionally, loop-mediated or recombinase polymerase isothermal assays have been designed for field deployment. The detection of MERS-CoV antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. Detection of viral proteins rather than viral RNA indicates the likely presence of infectious virus. The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity .",
"The first rapid immunochromatographic tool described could detect recombinant MERS-CoV nucleocapsid protein from DC nasal swabs with 94 % sensitivity and 100 % specificity compared to RT-rtPCR . A different approach used a monoclonal antibody-based capture ELISA targeting the MERS-CoV nucleocapsid protein with a sensitivity of 10 3 TCID 50 and 100 % specificity . Demonstration of a seroconversion to a MERS-CoV infection meets the current WHO definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior MERS-CoV infection and help support transmission studies. Because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral RNA . Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV.",
"Strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. Much of the Arabian Peninsula and all of the Horn of Africa lack baseline data describing the proportion of the community who may have been infected by a MERS-CoV. However, sero-surveys have had widespread use in elucidating the role of DCs as a transmission source for MERS-CoV. Because of the identity shared between DC and human MERS-CoV see Molecular epidemiology: using genomes to understand outbreaks , serological assays for DC sero-surveys should be transferrable to human screening with minimal re-configuration. Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction .",
"Also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested MERS-CoV isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single MERS-CoV serotype . The development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to MERS-CoV, as well as to likely sources of cross-reaction . Obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing . A number of commercial ELISA kits, immunofluorescent assays IFA kits, recombinant proteins and monoclonal antibodies have been released 31, . Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples .",
"Initially, conventional IFAs were used for human sero-surveys. These relied on MERS-CoV-infected cell culture as an antigen source, detecting the presence of human anti-MERS-CoV IgG, IgM or neutralizing antibodies in human samples . No sign of MERS-CoV antibodies was found among 2,400 sera from patients visiting Hospital in Jeddah, from 2010 through 2012, prior to the description of MERS-CoV . Nor did IFA methods detect any sign of prior MERS-CoV infection among a small sample of 130 healthy blood donors from another Hospital in Jeddah collected between Jan and Dec 2012 . Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA .",
"Of 226 slaughterhouse workers, only eight 3.5 % were positive by IFA, and those sera could not be confirmed by virus neutralization NT test. The study indicated that HCoV-HKU1 was a likely source of crossreactive antigen in the whole virus IFA . Whole virus MERS-CoV IFA also suffered from some cross-reactivity with convalescent SARS patient sera and this could not be resolved by an NT test which was also cross-reactive . IFA using recombinant proteins instead of whole-virus IFA, has been shown to be a more specific tool . Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead.",
"Since asymptomatic zoonoses have been posited , an absence of antibodies to MERS-CoV among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering DCs , a pre-existing cross-protective immune status or some other factor s resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. IFA using recombinant proteins instead. Some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the MERS-CoV nucleocapsid and spike proteins , or using a recombinant lentivirus expressing MERS-CoV spike protein and luciferase . A pseudo particle neutralization ppNT assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization MNT test. 10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay .",
"10, 74, Studies using small sample numbers and ppNT found no evidence of MERS-CoV neutralizing antibody in sera from 158 children with LRT infections between May 2010 and May 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the Jazan Region of the KSA during 2012 . Similarly, a study of four herdsmen in contact with an infected DC herd in Al-Ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in Al-Ahsa and 146 controls who were not exposed to DCs in any professional role, found none with serological evidence of past MERS-CoV infection using the ppNT assay . A delay in the neutralizing antibody response to MERS-CoV infection was associated with increased disease severity in South Korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks . The implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA.",
"A Jordanian outbreak of acute LRT disease in a hospital in 2012 was retrospectively found to be associated with MERS-CoV infection, initially using RT-rtPCR, but subsequently, and on a larger scale, through positivity by ELISA and IFA or MNT test. This outbreak predated the first case of MERS in the KSA. The ELISA used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-CoV HKU5 to identify antibodies against the equivalent crossreactive MERS-CoV protein . It was validated using 545 sera collected from people with prior HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-NL63, HRV, HMPV or influenza A H1N1 infections but was reportedly less specific than the recombinant IFA discussed above. It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing .",
"It was still considered an applicable tool for screening large sample numbers . A protein microarray expressing the S1 protein subunit has also been validated and widely used for DC testing . Detection of MERS-CoV infection using ELISA or S1 subunit protein microarray is usually followed by confirmatory IFA and/ or a plaque-reduction neutralization PRNT or MNT test. This confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in DCs bovine CoV, BCoV or humans HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV . In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result .",
"In the largest study of human sera, a tiered diagnostic process assigned both recombinant IFA and recombinant ELISA positive sera to 'stage 1' seropositivity. A stage 2 seropositive result additionally required a suitably titred PRNT result . The study found 15 sera collected in 2012 to 2013 from 10,009 0.2 % people in 13 KSA provinces contained MERS-CoV antibodies, but significantly higher proportions in occurred in camel shepherds two of 87; 2.3 % and slaughterhouse workers five of 140; 3.6 % . Contemporary surveys are needed. MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions.",
"MERS-CoV does not appear to be easily transmitted from DCs to humans, or perhaps it is , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. Serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. This was reinforced when a false positive US case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, NT assay and was subsequently retracted . The WHO recommends sampling from the LRT for MERS-CoV RT-rtPCR testing, especially when sample collection is delayed by a week or more after onset of symptoms. LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum .",
"LRT samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists . Recommended sample types include bronchoalveolar lavage BAL , tracheal/tracheobronchial aspirate, pleural fluid and sputum . Fresh samples yield better diagnostic results than refrigerated material and if delays in testing of ≥72 h are likely, samples except for blood should be frozen at −70°C . If available, lung biopsy or autopsy tissues can also be tested . The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR .",
"The URT is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when URT sampling is to be conducted . Paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting RT-rtPCR . Human urine and stool have been found to contain MERS-CoV RNA 12 to 26 days after symptom onset and are listed as samples that should be considered . In two cases that arrived in the Netherlands, urine was RT-rtPCR negative but faeces was weakly positive and sera were RT-rtPCR positive for five days or more . The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another .",
"The finding of MERS-CoV viral RNA in serum provides an avenue for retrospective PCR-based studies if respiratory samples are unavailable . RNAaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another . Clinically suspected MERS cases may return negative results by RT-rtPCR. Data have shown one or more negative URT samples may be contradicted by further URT sampling or the use of LRT samples, which is preferred . Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease .",
"Higher viral loads occur in the LRT compared to the URT. This fits with the observation that the majority of disease symptoms are reported to manifest as systemic and LRT disease . However, on occasion, even LRT specimens from MERS cases may initially be negative, only to later become positive by RT-PCR . This may be due to poor sampling when a cough is absent or non-productive or because the viral load is low . Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death .",
"Despite this both the largest human MERS-CoV studies 32, and smaller ones , use samples from the URT. It is then noteworthy that one study reported an association between higher loads in the URT and worse clinical outcome including intensive care and death . At writing, no human data exist to define whether the virus replicates solely or preferentially in the LRT or URT, or replicates in other human tissues in vivo although MERS-CoV RNA has been detected from both the URT and LRT in a macaque monkey model .The distribution of DPP4 in the human upper airways is also not well described. Individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others.",
"In one instance, a HCW shed viral RNA for 42 days in the absence of disease . It is an area of high priority to better understand whether such cases are able to infect others. Over three quarters of MERS cases shed viral RNA in their LRT specimens tracheal aspirates and sputum for at least 30 days, while only 30 % of contacts were still shedding RNA in their URT specimens . In the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates NPA; an URT sample , 30 tracheal aspirates, 13 sputa and three BAL were examined. The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death .",
"The tracheal aspirates and BAL returned the highest viral load values followed by NPA and sputum. Unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in NPA testing, were significantly correlated with severe disease and death . This study demonstrated the importance of LRT sampling for whole genome sequencing. When tested, samples positive for MERS-CoV are often negative for other pathogens . However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion .",
"However, many studies make no mention of additional testing for endemic human respiratory viruses . When viruses are sought, they have included human herpesvirus HHV , rhinoviruses HRV , enteroviruses EV , respiratory syncytial virus RSV , parainfluenzavirus types 1, 2 and 3 PIVs ,influenzaviruses IFVs , endemic HCoVs, adenoviruses AdVs metapneumovirus MPV and influenza A\\H1N1 virus; co-detections with MERS-CoV have been found on occasion . Bacterial testing is sometimes included for example, for Legionella and Pneumococcus but the impact of bacterial co-presence is also unclear 22, . Further testing of the LRT sample from the first MERS case used IFA to screen for some viruses negative for IFV, PIVs, RSV and AdVs and RT-PCR for others negative for AdV, EVs, MPV and HHVs . RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts.",
"RT-PCR also detected MERS-CoV. The WHO strongly recommends testing for other respiratory pathogens but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both MERS cases and their contacts. Little is known of other causes of MERS-like pneumonia in the KSA or of the general burden of disease due to the known classical respiratory viruses. Testing of adult pilgrims performing the Hajj in 2012 to 2014 has not detected any MERS-CoV. In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested .",
"In 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the KSA were tested . In 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested . It should be noted that most pilgrims arrived from MERS-free countries. A further 114 swabs were taken from pilgrims with influenza-like illness . In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims.",
"In earlier Hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. Over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among Hajj pilgrims. A LRT sample is often not collected for these studies , so false negative findings are a possibility although little is known about the initial site of MERS-CoV infection and replication; it may have been assumed it was the LRT because disease was first noticed there but the URT may be the site of the earliest replication. In Jeddah between March and July 2014 hereafter called the Jeddah-2014 outbreak; Fig. 3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % .",
"3 , there was a rapid increase in MERS cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 MERS-CoV detections ~3 % prevalence . Among 5,065 individuals sampled and tested across the KSA between October 2012 and September 2013,108 2.1 % detections were made in a hospital-centric population which included hospitalized cases n = 2,908; 57.4 % , their families n = 462; 9.1 % and associated HCWs n = 1,695; 33.5 % . Among the detections, 19 17.8 % were HCWs and 10 9.3 % were family contacts . The 2-3 % prevalence of active MERS-CoV infections is not dissimilar to the hospital-based prevalence of other human CoVs. However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\".",
"However, the proportion of deaths among those infected with MERS-CoV is much higher than that known for the HCoVs NL63, HKU1, 229E or OC43 in other countries, and even above that for SARS-CoV; it is not a virus that could reasonably be described as a \"storm in a teacup\". It is the low transmission rate that has prevented worldwide spread, despite many \"opportunities\". Very early in the MERS outbreak, some animals were highly regarded as either the reservoir or intermediate host s of MERS-CoV with three of the first five cases having contact with DCs . Today, animal MERS-CoV infections must be reported to the world organization for animal health as an emerging disease . A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease .",
"A summary of the first MERS cases reported by the WHO defined animal contact with humans as being direct and within 10 days prior to symptom onset . This definition made no specific allowance for acquisition from DCs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease . Camels are known to produce high levels of MERS-CoV RNA in their URT and lungs . Providing support for a droplet transmission route and perhaps indicating the presence of RNA in smaller, drier droplet nuclei, MERS-CoV RNA was identified in a high volume air sample collected from a barn housing an infected DC . The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 .",
"The precise source from which humans acquire MERS-CoV remains poorly studied but it seems likely that animal and human behavioural factors may play roles Fig. 3 . These factors may prove important for human cases who do not describe any DC contact nor any contact with a confirmed case. Whether the WHO definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals.",
"Wording focuses on consumption of DC products but does not specifically ascribe risk to a droplet route for acquisition of MERS-CoV from DC . Some MERS patients are listed in WHO disease notices as being in proximity to DCs or farms, but the individuals have not described coming into contact with the animals. No alternative path for acquiring infection is reported in many of these instances. What constitutes a definition of \"contact\" during these interviews has been defined for one study . Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 .",
"Despite this lack of clarity, the WHO consider that evidence linking MERS-CoV transmission between DCs to humans is irrefutable Fig. 4 . The possibility that bats were an animal host of MERS-CoV was initially widely discussed because of the existing diversity of coronaviruses known to reside among them . Conclusive evidence supporting bats as a source for human infections by MERS-CoV has yet to be found, but bats do appear to host ancestral representatives . However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event.",
"However, these are not variants of the same virus nor always within the same phylogenetic lineage as MERS-CoV; they are each a genetically distinct virus. Bat-to-human infection by MERS-CoV is a purely speculative event. The only piece of MERS-CoV-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the RNAdependent RNA polymerase gene of the MERS-CoV genome, identified in a faecal pellet from an insectivorous Emballonuridae bat, Taphozous perforatus found in Bisha, the KSA . While very short, the sequence of the fragment defined it as a diagnostic discovery. Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 .",
"Subsequently a link to DCs was reported and that link has matured into a verified association Fig. 4 . See figure on previous page. Fig. 3 Monthly detections of MERS-CoV blue bars and of cases who died red bars with some dates of interest marked for 2012 to 4 th September 2015. An approximation of when DC calving season and when recently born DCs are weaned is indicated. Spring green and summer orange in the Arabian Peninsula are also shaded. Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers .",
"Note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. Sources of these public data include the WHO, Ministries of Health and FluTrackers . Earlier and subsequent versions of this chart are maintained on a personal blog . Modified and reprinted from Mackay IM, Arden KE. Middle East respiratory syndrome: An emerging coronavirus infection tracked by the crowd. Virus Res 2015 Vol 202:60-88 with permission from Elsevier DCs, which make up 95 % of all camels, have a central presence in the Arabian Peninsula where human-DC contact ranges from little to close . Contact may be commonplace and could occur in variety of ways Fig. 4a .",
"Contact may be commonplace and could occur in variety of ways Fig. 4a . There are several large well-attended festivals, races, sales and parades which feature DCs and DCs are also kept and bred close to populated areas in the KSA . DC milk and meat are widely consumed and the older DC is an animal of ritual significance after the Hajj pilgrimage . However, MERS-CoV infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing DC products. Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits.",
"Daily ingestion of fresh unpasteurized DC milk is common among the desert Bedouin and many others in the KSA. DC urine is also consumed or used for supposed health benefits. Despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among MERS cases; this may simply be a reporting issue rather than an unexplainable absence of MERS. A small case-control study published in 2015 identified direct DC contact, and not ingestion of products, to be associated with onset of MERS . The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa .",
"The first sero-survey of livestock living in the Middle East region was conducted during 2012-2013 . DCs were sampled from a mostly Canary Island-born herd and from Omani DCs originally imported from the Horn of Africa . A neutralising antibody assay found only 10 % of strongly seropositive Canary Island . b Camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk of cases. There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody .",
"There are human MERS cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c Hypothetical ways in which subclinical when infection may not meet a previously defined clinical threshold of signs and/or symptoms or asymptomatic no obvious signs or measured, noticed or recalled symptoms of illness MERS-CoV infection may be implicated in transmission DC sera could neutralise MERS-CoV while all Omani DC sera had high levels of specific MERS-CoV neutralizing antibody . This indicated that DCs had in the past been infected by MERS-CoV, or a very similar virus. Since this study, a host of peer-reviewed reports have looked at both DCs and other animals, and the possibility that they may host MERS-CoV infection. Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV .",
"Seropositive DCs have been found throughout the Arabian Peninsula including Oman, the KSA, Qatar, Jordan, the United Arab Emirates UAE , Kuwait as well as Sudan, Somalia, Egypt, Tunisia, Nigeria, Kenya and Ethiopia in Africa and the Canary Islands 85, . Other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, Bactrian camels, llamas and guanaco south American camelids but none had detectable neutralising antibody against MERS-CoV . No virology or serology studies of human samples from areas in Africa where there are camels with a history of MERS-CoV have been reported to date. However,an absence of unexplained pneumonia that may be attributable to MERS-CoV infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, .",
"It is thus unclear whether MERS-CoV, or an antigenically related CoV, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the Arabian Peninsula . MERS-CoV RNA has also been detected in DC samples, and recovery of infectious virus has also been achieved from DC samples 4, 77, 117, 132, . From some of these, full or majority length genomes of MERS-CoV have been sequenced . DC versions of MERS-CoV were found to be as similar to each other, as were variants detected from different humans over time and across distance. Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV .",
"Antibody screening assays have also detected crossreactive antibodies in sera. These were identified as such by screening sera against similar viruses, for example BCoV or HCoV-OC43 as an antigenic facsimile for BCoV . It is possible that other MERS-CoV-like viruses also reside within DCs, but this does not detract from the definitive finding of MERS-CoV genetic sequences in both DCs and humans . Screening studies have shown that juvenile DCs are more often positive for virus or viral RNA while older DCs are more likely to be seropositive and RNA or virus negative . In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy .",
"In adult DCs, MERS-CoV RNA has been detected among animals with pre-existing antibody, suggesting re-infection is possible . Viral loads among positive DCs can be very high and DCs have been found positive both when ill with URT respiratory signs or when apparently healthy . These findings indicate DCs host natural MERS-CoV infections. Furthermore, stored DC sera have revealed signs of MERS-CoV in DCs which date back over three decades the earliest collected in 1983 . Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs?",
"Older sera have not been tested and so precisely how long DCs have been afflicted by MERS-CoV, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in Africa or the Arabian Peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. Researchers sought to determine a direction for infection; were DCs transmitting virus to humans or were humans infecting DCs? At a Qatari site, a farm owner and his employee became ill in mid-October 2013 and tested positive for MERS-CoV RNA in a sputum and throat swab sample, respectively. RT-rtPCRs found MERS-CoV RNA in 11 of 14 positive DC nasal swabs at the farm; six 43 % positive by two or more assays . The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences .",
"The results indicated a recent outbreak had occurred in this herd; the first indication of MERS-CoV RNA found within DCs with a temporal association to human infections. Three positive DC samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and DC MERS-CoV sequences . The DCs and human contacts yielded ORF1a and ORF4b sequences differing by only a single nucleotide each, clustering closely with the Hafr-Al-Batin_1_2013 variant . Subsequent case studies found evidence of a concurrent human and DC infection and the direction of that infection was inferred to be from the ill DCs and to their human owners . Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre .",
"Partial genome sequences indicated that a human and a MERS-CoV RT-rtPCR positive DC had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. All nine DC in the owner's herd, serially sampled, reacted in a recombinant S1 antigen ELISA, with the two animals that had been RT-rtPCR positive showing a small, verifiable rise in antibody titre . A rise in titre theoretically begins 10 to 21 days after DC infection . The authors suggested that the rise in titre in DC sera which occurred alongside a declining RNA load, while the patient was actively ill and hospitalized, indicated that the DCs were infected first followed by the owner . BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig.",
"BCoV antibodies were also present, and rising in one of the two RT-rtPCR positive animals but no animal's antibodies could neutralise BCoV infection . Camel calving season occurs in the winter months between late October and late February; Fig. 3 and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve DC populations . What role maternal camel antibody might play in delaying infection of calves remains unknown . Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, .",
"Juvenile DCs appear to host active infection more often than adult DCs and thus the sacrificial slaughter of DCs, which must be five years of age or older termed a thane , may not be accompanied by significant risk of exposure to infection. In contrast to earlier results, slaughterhouse workers who kill both younger and older DCs, may be an occupational group with significantly higher incidence of seropositivity to MERS-CoV when animals have active MERS-CoV infections 129, 139, . Expanded virological investigations of African DCs may lead to more seropositive animals and geographic areas in which humans may be at risk. It is possible that there are areas where humans already harbour MERS-CoV infections that have not been identified because of an absence of laboratory surveillance. Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h .",
"Virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures . Infectious MERS-CoV added to DC, goat or cow milk and stored at 4°C could be recovered at least 72 h later and, if stored at 22°C, recovery was possible for up to 48 h . MERS-CoV titre decreased somewhat when recovered from milk at 22°C but pasteurization completely ablated MERS-CoV infectivity . In a subsequent study, MERS-CoV RNA was identified in the milk, nasal secretion and faeces of DCs from Qatar . A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture.",
"A single study has examined the ability of MERS-CoV to survive in the environment . Plastic or steel surfaces were inoculated with 10 6 TCID 50 of MERS-CoV at different temperature and relative humidity RH and virus recovery was attempted in cell culture. At high ambient temperature 30°C and low RH 30 % MERS-CoV remained viable for 24 h . By comparison, a well known and efficently transmitted respiratory virus, influenza A virus, could not be recovered in culture beyond four hours under any conditions . Aerosol experiments found MERS-CoV viability only decreased 7 % at low RH at 20°C. In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV .",
"In comparison, influenza A virus decreased by 95 % . MERS-CoV survival is inferior to that previously demonstrated for SARS-CoV . For context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. MERS-CoV's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient . Whether MERS-CoV can remain adrift and infectious for extended periods truly airborne remains unknown. Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases.",
"Such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. The nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. Droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the Al-Ahsa hospital, the KSA and the South Korean outbreaks . By extrapolation, aerosol-generating events involving DCs urination, defecation, and preparation and consumption of DC products should be factored into risk measurement and reduction efforts and messaged using appropriate context. The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine.",
"The provision of evidence supporting the best formulation of personal protective equipment to be worn by HCWs who receive, manage or conduct procedures on infectious cases remains a priority. MERS-CoV was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. Sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to MERS-CoV transmission chains. However, transmission of MERS-CoV is defined as sporadic not sustained , intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact In a household study, 14 of 280 5 % contacts of 26 MERS-CoV positive index patients were RNA or antibody positive; the rate of general transmission, even in outbreaks is around 3 % . It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks.",
"It seems that the majority of human cases of MERS-CoV, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of MERS-CoV has not been self-sustaining . That is to say, the basic reproduction number R 0 -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. If R 0 was greater than 1, a sustained increase in case numbers would be expected. Some R o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan.",
"That MERS has had a constant presence in the Arabian Peninsula since 2012 is due to ongoing, sporadic spill-over events from DCs amplified by poorly controlled hospital outbreaks. The first known MERS human-to-human transmission event was one characterized by acute LRT disease in a healthcare setting in Jordan. In stark contrast, a sero-survey of HCW who were sometimes in close and prolonged contact with the first, fatal MERS-CoV case in 2012 , found none of the HCW had seroconverted four months later, despite an absence of eye protection and variable compliance with required PPE standards . Early on in the MERS story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases.",
"Contacts of confirmed MERS cases were often observed for clinical illness, but not tested. These omissions may have confounded our understanding of MERS-CoV transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. Case-control studies were not a focus. As testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized . A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill.",
"A rise in the cases termed asymptomatic which enlarge the denominator for calculations of the proportion of fatal cases, defined in resulted in a drop in the proportion of fatal cases during the Jeddah-2014 outbreak. Historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. Upon follow-up, over three-quarters of such MERS-CoV RNA positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic raising some question over the reliability of other reported data. The proportion of fatal MERS cases within the KSA compared to outside the KSA, as well as the age, and sex distribution change in different ways when comparing MERS outbreaks. Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die.",
"Approximately 43 % of MERS cases 549 of 1277 in the KSA were fatal betwen 2012 and December 2015 while 21 % 72 of 330 died among those occurring outside of the KSA. The total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. However the proportion of male deaths from total males with MERS is a similar figure to that for females. In the KSA, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 South Korean or the Jeddah-2014 outbreaks Additional file 2: Figure S2 . Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea.",
"Why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected habits, exposure to a human or zoonotic source, viral load, route of infection or the extent to which different populations are burdened by underlying diseases . As a group, HCWs comprised 16 % of MERS cases in the KSA and South Korea. It is apparent that the weekly proportion of infected HCWs increases alongside each steep rise in overall detections Fig. 5 . In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks .",
"In May 2013, the WHO published guidelines for IPC during care of probable or confirmed cases of MERS-CoV infection in a healthcare setting . This is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks . These rises in MERS-CoV detections can decrease the average age during each event because HCWs are usually younger than inpatients with MERS. Healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control IPC procedures . Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach.",
"Most of the analysis of MERS-CoV genetics has been performed using high throughput or \"deep\" sequencing methods for complete genome deduction . MERS-CoV was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. The technique can produce genomic . Earlier and subsequent versions of this chart are maintained on a personal blog length coverage in a single experiment with highly repetitious measurement of each nucleotide position . Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig.",
"Despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during MERS-CoV characterization . As more genomes from both humans and DCs have been characterized, two clades have become apparent; A and B Fig. 6 . Clade A contains only human-derived MERS-CoV genomes from Jordan, while Clade B comprises the majority of human and camel genomes deduced thus far . Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur .",
"Two studies during 2015, one looking at Jeddah-2014 MERS-CoV variants and another looking at a variant exported from South Korea to China, have now identified signs of genetic recombination among MERS-CoV variants. While human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur . Shared identity implies that the major source for human acquisition is the DC, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. Over a month, a DC virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that DCs are the natural, rather than intermediate, host for the MERS-CoV we know today . To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 .",
"To date, recombination has been localised to breakpoints near the boundary between ORF1a and ORF1b regions, within the spike gene and in the ORF1b region Fig. 2 . It is not unexpected that recombination should occur since it is well known among other CoVs and because the majority of MERS-CoV whole genomes collected from samples spanning three years 2012-2015 and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied 52, 77, 135, 138, 168, . Changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org .",
"If we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely Fig. 6 The genetic relationship between MERS-CoV nucleotide sequences downloaded from GenBank using the listed accession numbers and from virological.org . This neighbour joining tree was created in MEGA v6 using an alignment of human and DCderived MERS-CoV sequences Geneious v8.1 . Clades are indicated next to dark Clade A or pale Clade B blue vertical bars. Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur.",
"Camel icons denote genomes from DCs. Healthcare or community outbreaks are boxed and labelled using previously described schemes monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. Genetic mutations noted during the largest of human outbreaks, Jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro . However, we understand very little of the phenotypic outcomes that result from subtle genetic change in MERS-CoV genomes. To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed.",
"To date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses . But vigilance and larger, more contemporary and in vivo studies are needed. Genome sequence located to a distinct clade were identified from an Egyptian DC that was probably imported from Sudan. This does not fit into either of the current clades . A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig.",
"A virus sequenced from a Neoromicia capensis bat was more closely related to MERS-CoV than other large bat-derived sequences had been to that point, but the genome of a variant of a MERS-CoV has yet to be discovered and deduced from any bat . Analyses of MERS-CoV genomes have shown that most single nucleotide differences among variants were located in the last third of the genome Fig. 2 , which encodes the spike protein and accessory proteins . At least nine MERS-CoV genomes contained amino acid substitutions in the receptor binding domain RBD of the spike protein and codons 158 N-terminal region , 460 RBD , 1020 in heptad repeat 1 , 1202 and 1208 bear investigation as markers of adaptive change . The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants.",
"The spike protein had not changed in the recombinant MERS-CoV genome identified in China in 2015 but was reported to have varied at a higher rate than that for complete MERS-CoV genomes, among South Korean variants . This highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. Despite this, one assay amplifying a 615 nucleotide fragment of the spike S2 domain gene for Sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings . Genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times Fig. 6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA.",
"6 . This approach was employed when defining the geographically constrained MERS hospital outbreak in Al-Ahsa, which occurred between 1 st April and 23 rd May 2013, as well as clusters in Buraidah and a community outbreak in Hafr Al-Batin, the KSA. Genomic sequencing identified that approximately 12 MERS-CoV detections from a community outbreak in Hafr Al-Batin between June and August 2013 may have been triggered by an index case becoming infected through DC contact . Sequencing MERS-CoV genomes from the 2013 Al-Ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare .",
"Molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months . However, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare . In Riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous . Riyadh is a nexus for camel and human travel and has had more MERS cases than any other region of the KSA to date but also harbours a wide range of MERS-CoV variants . However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation .",
"However the South Korean outbreak originated from a single infected person, resulting in three to four generations of cases . Studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation . For many MERS cases detected outside the Arabian Peninsula, extensive contact tracing has been performed and the results described in detail. Contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event.",
"Although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. Results of contact tracing to date have found that onward transmission among humans is an infrequent event. For example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in Germany who travelled from the UAE but no sign of virus or antibody were found in any of them . The very first MERS case had made contact with 56 HCWs and 48 others, but none developed any indication of infection . In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive .",
"In a study of 123 contacts of a case treated in France, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive . None of the contacts of the first two MERS cases imported into the USA in 2014 contained any MERS-CoV footprint and none of the 131 contacts of two travellers returning to the Netherlands developed MERS-CoV antibodies or tested RNA positive . Analyses of public data reveal many likely instances of nosocomial acquisition of infection in the Arabian Peninsula and these data may be accompanied by some details noting contact with a known case or facility. One example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster . Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig.",
"Contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple South Korean hospitals . Such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease Fig. 4c . The hospital-associated outbreak in Jeddah in 2014 was the largest and most rapid accumulation of MERS-CoV detections to date. The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control .",
"The greatest number of MERS-CoV detections of any month on record occurred in Jeddah in April. The outbreak was mostly >60 % of cases associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control . A rise in fatalities followed the rapid increase in case numbers. In 2015 two large outbreaks occurred. South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November.",
"South Korea was the site of the first large scale outbreak outside the Arabian Peninsula and produced the first cases in both South Korea and China, occurring between May and July 2015. This was closely followed by a distinct outbreak in Ar Riyad province in the KSA which appeared to come under control in early November. After staying in Bahrain for two weeks, a 68 year old male 68 M travelled home to South Korea via Qatar, arriving free of symptoms on the 4 th May 2015 . He developed fever, myalgia and a cough nearly a week later 11 th . He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th .",
"He visited a clinic as an outpatient between the 12 th and 15 th of May and was admitted to Hospital A on the 15 th . He was discharged from Hospital A on the 17 th then visited and was admitted to the emergency department of Hospital B on the 18 th . During this second stay, a sputum sample was taken and tested positive for MERS-CoV on the 20 th , triggering transfer to the designated isolation treatment facility. Over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of \"hospital shopping\" that shaped the South Korean outbreak. Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died .",
"Approximately 34 people were infected during this time . In total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 M; 37 cases died . In South Korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them . Other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with MERS, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of Korean society, crowded emergency rooms, poor IPC measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories 24, . All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal.",
"All of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired 24, 120, 181, . Few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. The hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact 191, . Early in the outbreak, a infected traveller, the son of an identified case in South Korea, passed through Hong Kong on his way to China where he was located, isolated and cared for in China . No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased .",
"No contacts became ill. The outbreak was brought under control in late July/ early August after improved IPC measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased . A review of public data showed that, as for MERS in the KSA, older age and the presence of underlying disease were significantly associated with a fatal outcome in South Korea. Even though R 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from MERS-CoV infection . The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 .",
"The dynamic of an outbreak depends on the R 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density . In the region of Ar Riyad, including the capital city of Riyadh, a hospital based cluster began, within a single hospital, from late June 2015 . By mid-September there had been approximately170 cases reported but the outbreak appeared to been brought under control in November. It became apparent early on that MERS-CoV spread relatively ineffectively from human-to-human. Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality.",
"Despite ongoing and possibly seasonal introduction of virus to the human population via infected DCs and perhaps other animals yet to be identified, the vast majority of MERS-CoV transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. This opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. It remains unclear if this group are uniquely affected by MERS-CoV or if other respiratory virus infections, including those from HCoVs, produce a similarly serious impact. In South Korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting.",
"Household human-to human transmission occurs but is also limited. Educational programs will be essential tools for combatting the spread of MERS-CoV both within urban and regional communities and for the health care setting. Vigilance remains important for containment since MERS-CoV is a virus with a genetic makeup that has been observed for only three years and is not stable. Among all humans reported to be infected, nearly 40 % have died. Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers.",
"Continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. Global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. Whole genome sequencing has been used extensively to study MERS-CoV travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. MERS and SARS have some clinical similarities but they also diverge significantly . Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV.",
"Defining characteristics include the higher PFC among MERS cases above 50 % in 2013 and currently at 30-40 %; well above the 9 % of SARS and the higher association between fatal MERS and older males with underlying comorbidities. For the viruses, MERS-CoV has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to SARS-CoV. There appears to be a 2-3 % prevalence of MERS-CoV in the KSA with a 5 % chance of secondary transmission within the household. There is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any R 0 would predict on face value. Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern.",
"Nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of MERS or MERS-CoV during or immediately after these events. There is no evidence that MERS-CoV is a virus of pandemic concern. Nonetheless, hospital settings continue to describe MERS cases and outbreaks in the Arabian Peninsula. As long as we facilitate the spread of MERS-CoV among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic.",
"The MERS-CoV appears to be an enzootic virus infecting the DC URT with evidence of recent genetic recombination. It may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. Thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. RT-PCR testing of LRT samples remains the gold standard for MERS-CoV confirmation. Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered.",
"Serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. Similarly, the important question of whether those who do shed MERS-CoV RNA for extended periods are infectious while appearing well, continues to go unanswered. It is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. While the basic virology of MERS-CoV has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. Additional file 1: Figure S1 . The"
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